Rab7l1 interacts with lrrk2 to modify intraneuronal protein sorting and parkinson&#39;s disease risk

ABSTRACT

The present invention relates to methods and compositions for treating Parkinson&#39;s Disease in a subject.

This application is a continuation-in-part of U.S. application Ser. No. 14/760,421 filed Jul. 10, 2015, which is a U.S. National Phase application under 35 U.S.C. §371, of International Application No. PCT/US2014/011226 filed on Jan. 13, 2014, which claims priority to U.S. Application Ser. No. 61/751,435 filed Jan. 11, 2013, the contents of which are hereby incorporated by reference in their entireties.

GOVERNMENT INTERESTS

The work described herein was supported in whole, or in part, by National Institute of Health Grant Nos. NS064433 and NS060876. The United States Government has certain rights to the invention.

All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND

Parkinson's disease (PD) is a degenerative disorder of the central nervous system. It results from the death of dopamine-containing cells in the substantia nigra, a region of the midbrain; the cause of cell-death is unknown. Early in the course of the disease, the most obvious symptoms are movement-related, including shaking, rigidity, slowness of movement and difficulty with walking and gait. Later, cognitive and behavioral problems may arise, with dementia commonly occurring in the advanced stages of the disease. Other symptoms include sensory, sleep and emotional problems. PD is more common in the elderly with most cases occurring after the age of 50.

Parkinson's disease is diagnosed by a physician exam, and diagnosis is based on the medical history and a neurological examination of the patient. There is no laboratory or molecular test that will clearly identify the disease. Brain scans are sometimes used to rule out disorders that could give rise to similar symptoms. Patients may be given levodopa, or other dopamine affecting agent, and resulting relief of motor impairment tends to confirm diagnosis. The finding of Lewy bodies in the midbrain on autopsy is usually considered proof that the patient suffered from Parkinson's disease. Thus, there is need for biomarkers for PD disease or treatment.

SUMMARY

In certain aspects the invention provides methods for treating Parkinson's Disease (PD), comprising administering to a subject in need thereof a therapeutic amount of a retromer complex-stabilizing compound, or a pharmaceutically acceptable salt thereof. In some embodiments, the retromer complex-stabilizing compound stabilizes VPS35, VPS29, VPS26 or a combination thereof. In some embodiments, the retromer complex-stabilizing compound stabilizes the interaction between VPS35 and VPS29. In some embodiments, the retromer complex-stabilizing compound is a compound of formula (I),

wherein Ar is a 5- or 6-membered aromatic ring or a 5- or 6-membered heteroaromatic ring having 1-4 heteroatoms independently selected from sulfur, nitrogen, or oxygen; X is

or H; R¹ is C₁-C₆-alkyl; A is —S—R², —S(O)R², —SO₂R², —SO₃R²,

and each R² is independently selected from H, C₁-C₆-alkyl, or phenyl. In some embodiments, the compound of formula (I) binds to VPS35 and VPS29. In some embodiments, the retromer complex-stabilizing compound is R55, wherein the structure of R55 is

In some embodiments, the retromer complex-stabilizing compound is R33, wherein the structure of R33 is

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating PD in a subject. In one embodiment, the method comprises measuring the expression levels of a set of genes in a sample from a subject, wherein the set of genes comprises at least one gene selected from the genes listed in Table 2; comparing the expression levels of the set of genes in the subject sample to expression levels of the same set of genes in a reference sample or samples, wherein the reference sample or samples are from an individual who has a PD-associated SNP, and wherein similar expression levels of the set of genes in the subject sample and the set of genes in the reference sample(s) indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides a method of treating PD in a subject. In one embodiment, the method comprises determining a level of full-length RAB7L1 in a sample from a subject; comparing the level of full-length RAB7L1 from the subject sample to a full-length RAB7L1 level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the full-length RAB7L in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the method comprises the level of full-length RAB7L is protein level of full-length RAB7L, or mRNA levels of the full-length RAB7L, or a combination thereof. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining a level of isoform 3 of RAB7L1 in a sample from a subject; comparing the level of isoform 3 of RAB7L1 from the subject sample to an isoform 3 of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of isoform 3 of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In another embodiment, the level of isoform 3 of RAB7L1 is a protein level. In a further embodiment, the method further comprises determining the level of transcript variant 4, 5, or a combination thereof of RAB7L1. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining a level of transcript variant 4, 5, or a combination thereof of RAB7L1 in a sample from a subject; comparing the level of transcript variant 4, 5, or a combination thereof of RAB7L1 from the subject sample to a transcript variant 4, 5, or a combination thereof of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of transcript variant 4, 5, or a combination thereof of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In another embodiment, the level of transcript variant 4, 5 or a combination thereof of RAB7L1 is a mRNA level. In a further embodiment, the method further comprises determining the level of isoform 3 of RAB7L1. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining a level of retromer components in a sample from a subject; comparing the level of retromer components from the subject sample to a retromer component level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the retromer components in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In another embodiment, the level of retromer component is protein level of retromer component, or mRNA levels of retromer component, or a combination thereof. In a further embodiment, the retromer component is VPS35, VPS29, VPS26 or a combination thereof. In some embodiments, the level of VPS35, VPS29, or VPS26 is protein level of VPS35, VPS29, or VPS26, or mRNA levels of VPS35, VPS29, or VPS26, or a combination thereof. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides for a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of at least two different markers in a subject sample, wherein the markers comprise LRRK2, RAB7L1 and VPS35.

An aspect of the invention provides for a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of a different marker in a subject sample, each marker being one of the genes listed in Table 2. In one embodiment, the composition comprises a microarray, a microfluidics card, a chip, or a chamber.

An aspect of the invention provides a kit for determining the levels of RAB7L1, LRRK2, VPS35, or a combination thereof, the kit comprising at least one oligonucleotide or polynucleotide to selectively quantify the levels of RAB7L1, LRRK2, VPS35, or a combination thereof. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 15, 16, 17, or 18.

An aspect of the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence or absence of a PD-associated genetic variant, the kit comprising at least one oligonucleotide or polynucleotide for sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 24, or 25.

An aspect of the invention provides for a diagnostic kit comprising the microarray, microfluidics card, chip, or chamber described herein.

An aspect of the invention provides for a synthetic nucleic acid comprising SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one color drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee. The original color versions of FIGS. 1-13 can be viewed in MacLeod et al., (2013) Neuron. 77(3):425-39 (including the accompanying Supplementary Information available in the on-line version of the manuscript available on the Neuron web site). The contents of MacLeod et al., (2013) Neuron. 77(3):425-39, including the accompanying “Supplementary Information,” are herein incorporated by reference.

FIGS. 1A-1B. LRRK2 and PARK16 PD risk-associated variants function in a common genetic pathway. (1A) PD risk-associated variants exert functional effects in the CNS of unaffected individuals that is assessed in terms of a global transcriptome impact. Similar to the one observed in PD affected brain, it may reflect a pre-disease prodromal state. (1B) Schematic of GPI analysis. Without being bound by theory, PD risk-associated genotypes at 2 independent loci (upper panels) differentially alter the function of a nearby gene (red star in middle panel). This secondarily impacts the brain transcriptome (lower panels), with significant overlap for different PD-risk genotype shows.

FIGS. 1C-1D. LRRK2 and PARK16 PD risk-associated variants function in a common genetic pathway. (1C) Hierarchical clustering dendrogram shows that the gene expression signatures across 7 PD-associated variant GPIs (“Risk GPI”; in unaffected cerebral cortex Broadmann Area 9 [BA9]) are most similar to the signatures seen in PD brain (BA9 or substantia nigra; SN; in red (e.g., PD/S.N. and PD/Cx.) rather than in other CNS diseases such as Alzheimer's disease, Huntington's disease, Bipolar Disorder or Schizophrenia. 352 gene transcript expression patterns—corresponding to the intersection of the PD risk variants GPIs (FIG. 8A-C)—were interrogated. Clustering was performed using Pearson's distance with complete linkage (see Methods). (1D) Genetic interaction between PARK16 and LRRK2 alleles revealed by GPI analysis in 185 unaffected brain samples (GEO GSE15222 “Initial”) and in an independent cohort of 143 unaffected brain samples (GEO GSE15745, “Replication”), as established by the interaction factor between pairs of GPIs as indicated, in a linear regression model (see Methods). The p-value (“p”) associated with the interaction term as well as its orientation (“Dir.”) are presented. Results combined across both cohorts presented (“Combined”) with the resulting Z-scores and p-values for interaction.

FIGS. 1E-1F. LRRK2 and PARK16 PD risk-associated variants function in a common genetic pathway. (1E) The PARK16 genotype modifies LRRK2 associated risk in sporadic PD. A table presents the odds ratios for PD at the LRRK2 locus as a function of the PARK16 genotype in 4 independent GWAS cohorts: 1 of Ashkenazi Jews (“AJ”, n=417) and 3 of Caucasians (“NGRC”, n=4008; “NINDS”, n=537; “MAYO”, n=886). (1F) Manhattan plot of the Chr1 region reported as a modifier of age of disease onset in familial PD with LRRK2 mutation (Latourelle et al., 2011). Epistasis was evaluated for 74 SNPs in 4 independent sporadic PD GWAS datasets. X-axis represents chromosomal location, Y-axis represents −log 10 of the combined p-value for epistasis of each SNP with the PD risk SNP rs11176052 at the LRRK2 locus. The PARK16 locus PD-associated SNP rs823114 (arrow) exhibited the most significant association (p=4.6 E-6; red line (shown as grey in black and white image) represents the significance threshold after correction for multiple testing).

FIG. 2A shows schematics of the PARK16 locus on chromosome 1.

FIG. 2B. Overexpression of the PARK16 locus gene RAB7L1 specifically rescues a LRRK2 mutant phenotype. RAB7L1 modifies a LRRK2-associated neurite process length phenotype. Rat primary cortical neuron cultures transfected with a vector expressing G2019S mutant LRRK2 displayed reduced total neurite length relative to vector alone (cells are co-transfected with GFP for visualization by fluorescence microscopy). (i) (ii) Co-transfection of a wild-type or constitutively active (CA) RAB7L1 expression vector (1 μg/well) along with LRRK2 G2019S (0.5 μg/well) significantly rescued neurite length; other PARK16 genes—NUCKS1, SLC45A3, PM20D1, and SLC41A1—failed to rescue. CA or inactive (IN) RAB vectors were also tested as indicated (left panel; GFP-tagged at the N-terminus; 1 μg/well). (iii) Knock-down of RAB7L1 by shRNA vector transfection led to a similar decrease in neurite length as with LRRK2 G2019S expression. n=20 neurons in 4 independent cultures per group. Mean total neurite lengths are displayed; error bars represent SEM, *: p<0.05, **: p<0.01, ***: p<0.001 for ANOVA followed by Tukey's HSD post hoc analysis.

FIGS. 3A-3C. Evidence of a RAB7L1-LRRK2 complex. (3A) Immunoprecipitation (IP) analysis of RAB7L1 from lysates of HEK293T cells transfected with plasmids encoding a GFP-RAB7L1 fusion protein (or vector alone) and a 3×flag (3FL) epitope-tagged LRRK2 construct (either wild type [WT], G2019S [GS], K1906M [KM], or empty vector). IP with an anti-flag antibody was followed with immunoblot (IB) analysis with an anti-GFP or an anti-LRRK2 antibody as indicated. Arrowheads indicate the expected protein sizes. (3B) Co-immunoprecipitation of LRRK2 with RAB7L1 from lysates of HEK293T cells transfected with a plasmid encoding a 3×flag LRRK2 construct and a plasmid encoding a GFP-RAB7L1 fusion protein (either WT, CA, IN, or GFP only). (3C) Immunoprecipitation using an anti-LRRK2 antibody from whole brain lysates of non-transgenic (NT), LRRK2 wild type transgenic (WT), LRRK2 R1441C (RC) transgenic, or LRRK2 knockout (−/−) mice. TB was subsequently performed for RAB7L1, RAB11 and LRRK2.

FIG. 3D. Evidence of a RAB7L1-LRRK2 complex. (3D) Subcellular co-localization of RAB7L1 and LRRK2. Human neuroblastoma SH-SY5Y cells were transfected with GFP-tagged RAB7L1 vectors (in green (shown as light grey in black and white image)); either WT, CA, or IN forms, as well as a RAB7L1 construct lacking exon 2 and 3 and corresponding to an alternatively spliced RAB7L1 transcript, “AT”) and a 3×flag-tagged LRRK2 vector (in red, left panel (shown as grey in black and white image)). Subcellular localization was determined by immunostaining with a marker for the Golgi apparatus (Golph4; in blue (shown as dark grey in black and white image)). The CA form leads to a reduced localization to the Golgi apparatus. Co-localization is evaluated by quantifying the fraction of RAB7L1/Golph4, RAB7L1/LRRK2 and LRRK2/Golph4 staining overlap (Upper, lower and middle right panels respectively). Results represent mean±SEM (n=15 per group).

FIG. 4A. RAB7L1 rescues lethality and dopamine neuron loss in a Drosophila model of LRRK2 G2019S neurodegeneration. Modifier screen for suppressors of an early adult lethality phenotype seen with expression of LRRK2 G2019S selectively in tyrosine hydroxylase (TH)-positive dopamine neurons. Left, a panel of 16 Drosophila RAB transgenes was screened (of 31 total; see Table 3). Adult survival (days post-eclosion) curves are presented for individual strains harboring different RABs along with the LRRK2 G2019S transgene. Non-transgenic survival curve is shown for comparison. n>25 for all conditions. Right, Adult survival (days post-eclosion) of Drosophila is presented in the context of transgenic expression of LRRK2 (WT or G2019S), with or without RAB7L1 (WT, CA or IN), using a tyrosine hydroxylase promoter GAL4 driver for dopaminergic neuron expression. Non-transgenic survival is also shown for comparison. n>25 for all conditions.

FIG. 4B. RAB7L1 rescues lethality and dopamine neuron loss in a Drosophila model of LRRK2 G2019S neurodegeneration. (left) Confocal microscopy of mushroom bodies of the CNS from transgenic Drosophila as in (FIG. 4A), with dopaminergic neuron nuclei visualized using an additional marker transgene, a nuclear localization sequence (NLS)-GFP fusion, also driven by TH-Gal4. (Right) Quantitation of surviving dopaminergic neurons in the PPM1 and PPL1 clusters of Drosophila CNS mushroom bodies. Means are displayed; error bars represent SEM; ***: p<0.001 by ANOVA followed by Tukey's HSD post hoc analysis for (4A) and (4B).

FIGS. 5A-5D. PARK16 PD risk-associated variants modify RAB7L1 splicing and protein accumulation. (5A) Exonic structure of the human RAB7L1 gene. (5B) Analysis of RAB7L1 alternative splicing in human cortical brain samples. The rs1572931 allele G, linked to the PD high-risk haplotype (R), is associated with an increase in the fraction of RAB7L1 transcripts that lack the exon 2 to exon 3 junction region (termed exon 2 skipping; presented relative to the extent of exon 2 skipping seen in carriers of the rs1572931 protective allele A; quantified by qrtPCR using primers as depicted by red and black arrows in (5A) detecting respectively the amount of total and unskipped RAB7L1 mRNA; n=15 and 57 for P and R respectively; details in Table 6). (5C) (i) Schematic of predicted splice site enhancer and silencer motifs (SEQ ID NOS 29 and 30 respectively, in order of appearance) upstream of RAB7L1 exon2 and affected by rs1572931 variants G (associated with increased PD risk, “R”) and A (protective, “P”, associated with decreased PD risk). (ii) Structure of a minigene construct to assess the effect of rs1572931 variants on RAB7L1 exon2 inclusion in vitro. Green arrows indicate the position of the primers used to assess exon 2 inclusion. (5D) Impact of PARK16 variants on splicing in vitro. The rs1572931 allele G (associated with increased PD risk, R; relative to the allele A associated with decreased PD risk, P) leads to a relative decrease in RAB7L1 exon 2 inclusion in transfected human SH-SY5Y cells as assess by PCR gel quantification (pictures in FIGS. 12D-E; n=6/group).

FIG. 5E is a bar graph showing the impact of rs1572931 on RAB7L1 protein level in human cortical brain samples. rs1572931 allele G is associated with a decrease in RAB7L1 protein level in non-PD post-mortem human cortical brain samples, as assessed by Western Blot from individuals homozygous for the risk allele (R, n=25) and from carriers of the protective allele (P, n=13). Mean levels are displayed; errors bars are SEM; *: p<0.05, **: p<0.01, ***: p<0.001 by two-tailed t-test (5B, 5D) or by linear regression analysis (5E).

FIGS. 6A-6C. RAB7L1 and LRRK2 modulate lysosome and Golgi apparatus sorting in a retromer-dependent manner. (6A-6C) Analysis of MPR sorting in primary rat neuron cultures transfected with vectors encoding LRRK2 G2019S mutant (GS), RAB7L1, VPS35, or VPS35 D620N; or with shRNA plasmids for VPS35, RAB7L1 or vector only, co-transfected with GFP vector for visualization and immunostained for MPR as well as either the Golgi marker Golph4 (6A, upper panel), the lysosome marker Lamp2 (6B, upper panel) or with the early endosome marker EEA1 (6B, upper panel). MPR colocalization with either the Golph4 or LAMP2 marker was reduced with G2019S LRRK2, VPS35 D620N, or knockdown of either RAB711 or VPS35 (6A, lower panel; 6B, lower panel). These manipulations also increased total LAMP2 staining (but not Golph4 staining). Scale bar represents 10 um. Quantifications of the MPR co-localization and of total organelle marker analyses are presented in the lower panels. Error bars represent SEM. n>10 cells in 3 independent wells per group. *: p<0.05, **: p<0.01, ***: p<0.001 for comparisons with “vector” group, ++: p<0.01, +++: p<0.001 for comparisons with “LRRK2 G2019S” group by ANOVA followed by Tukey's HSD post hoc analysis.

FIG. 6D is a schematic showing cell sorting phenotype associated with defects in the LRRK2-Rab7L1 pathway or knockdown of the VPS35 retromer component. MPR accumulation at Golph4-positive structures (trans-golgi network [TGN]) and at LAMP2-positive structures (lysosomes and late endosomes [LE]) is reduced, and lysosomes appear swollen.

FIG. 7A. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. Transfection of rat primary cortical neuron cultures with a wild-type (WT) VPS35 expression vector rescued the reduced neurite length phenotype associated with LRRK2 G2019S (GS) mutant expression or with Rab7L1 (R7L1) knockdown. Overexpression of a familial PD mutant VPS35 D620N vector leads to reduced neurite length relative to vector alone. Knockdown of VPS35 by shRNA leads to similarly reduced neurite length relative to vector alone, which is not rescued by Rab7L1 overexpression (n=20 neurons in 4 cultures per group).

FIG. 7B. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. (Left) Confocal microscopy of mushroom bodies of the CNS from transgenic Drosophila with dopaminergic neuron nuclei visualized using a TH-Gal4-driven nuclear localization sequence (NLS)-GFP fusion. (Right) Quantitation of surviving dopaminergic neurons in the PPM1 and PPL1 clusters of Drosophila CNS mushroom bodies.

FIG. 7C. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. Relative quantification by western blot of VPS35 (left) or VPS29 (right) protein levels in lysates from mouse neuroblastoma (N2a) cells transfected with vectors encoding VPS35 WT, VPS35 shRNA, VPS35 D620N, LRRK2 WT, LRRK2 G2019S (GS), RAB7L1, RAB7L1 shRNA, or vector control (N=3/group).

FIG. 7D. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. LRRK2 impacts the levels of retromer components in mouse brain. Relative quantification by Western blotting of VPS35 (left), VPS29 (middle) and VPS26 (right) levels in brain tissue samples from non-transgenic (“nTg”), LRRK2 wild-type (“LRRK2-WT”) and LRRK2 R1441C mutant (“LRRK2-RC”) BAC transgenic mice (N=3/group).

FIG. 7E. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. Immunoprecipitation (IP) analysis of RAB7L1 from lysates of SH-SY5Y cells transfected with plasmids encoding a GFP-VPS35 fusion protein with VPS35 wild-type sequence (“WT”) or the familial PD mutant D620M (“D620N) or vector alone, along with a LRRK2 construct or an empty vector. IP with an anti-GFP antibody was followed with Western immunoblot analysis with an anti-LRRK2 or anti-GFP antibody as indicated. Arrowheads indicate the expected protein sizes.

FIG. 7F. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. IP using an anti-LRRK2 antibody from whole brain lysates of non-transgenic (NT), LRRK2 wild type transgenic (WT), LRRK2 R1441C (RC) transgenic, or LRRK2 knockout (−/−) mice as in FIG. 3D. Immunoblot was subsequently performed for VPS35 and β-Actin.

FIG. 7G. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. VPS35 mRNA levels in substantia nigra tissue as determined by meta-analysis of 5 gene expression microarray datasets (Table 5) in 63 unaffected individuals and 81 PD patients samples (left panel) and in laser-microdissected substantia nigra dopaminergic neurons from 8 unaffected individuals and 10 PD patients samples (right panel, GEO GSE20141). Expression levels are normalized to mean of the unaffected group.

FIGS. 7H-7I. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. (H) VPS35 mRNA in cerebral cortex tissue as determined by high-throughput sequencing of the 3′UTR ends of polyadenylated mRNA transcripts on a cohort of 17 unaffected and 17 PD cerebral cortical tissue samples. Levels are expressed as reads per million (rpm). (I) VPS35 mRNA levels in Globus Pallidus Interna (Gpi) samples (n=10/group GEO GSE20146). Expression levels are normalized to mean of the unaffected group. For all graphs means are displayed, error bars represent SEM; p<0.05(*) p<0.01(**) for ANOVA followed by Tukey's HSD post hoc analysis (7A, 7B, 7C) or by two-tailed t-test (7G, 7H).

FIG. 8A is a graph depicting GPIs of SNPs at 7 PD-associated loci that show a high degree of overlap. Overlap was quantified in terms of number of gene expression profiles that were impacted in the same direction by 7 disease risk-associated haplotypes (at the SNCA, MAPT, LRRK2, PARK16, HLA-DRA, STK39 and LAMP3 loci). 352 were impacted in the same orientation (up or down, of 8560 queried) by all 7 SNPs. Such overlap is highly significant, as shown by analysis of randomly chosen sets of SNP variants. Analysis of 25000 randomly chosen sets of 7 SNPs is shown for comparison, with average of 19.5 overlapping genes and count distribution as shown. p=1E-5 using Wald statistics (see Methods).

FIG. 8B is a histogram of the resampling result for the estimation of the significance between the PD-risk GPI and the expression profile characteristic of PD in prefrontal cortex.

FIG. 8C is a schematic that shows the correlation pattern for each of the genes belonging to the PD-risk intersection GPI with a FDR<5% and that also shows a significant difference (p<0.05, two tailed t test) in their expression levels in either BA9 or SN for a PD vs unaffected comparison.

FIGS. 8D-8E shows Gene Ontology categories enriched in genes whose expression levels are positively (red (first 5 rows)) or negatively (blue (last 5 rows)) associated with the PD risk-associated allelic load for all PD loci (8D) and specifically for the LRRK2 and PARK16 loci (8E). Analysis were conducted using DAVID.

FIG. 9A is a bar graph depicting total neurite length of rat primary cortical neurons transfected with vector or LRRK2 WT or LRRK2 R1441C (0.5 ug per well) and RAB7L1 expressing vector or empty vector (1 ug per well) as indicated. Means are represented, errors bars are SEM. N=20 per group.

FIGS. 9B-9C. Rab7L1 knockdown efficiency measured by Western blot quantitation in 3 independent vector or Rab7L1 shRNA transfected cultures (9B). Graph shows relative band intensity+/−SEM*p<0.05 by two-tailed t-test. Validation of PARK16 locus genes overexpression vectors by Western Blot (9C). Lysates from cells transfected with the PARK16 gene indicated (+) or control vector (−) probed by immunoblot using corresponding antibodies that recognize both endogenous and exogenous PARK16 gene expression. Constructs were transfected in cell lines of matching species (human SH-SY5Y for RAB7L1 and NUCKS1; mouse N2a for SLC41A1 and SLC45A3). 30 ug protein was loaded per lane. Beta-actin loading control is shown below.

FIG. 10A is a photographic image of Rab7L1 and LRRK2 immunohistochemistry of substantia nigra section from non-transgenic, LRRK2 WT, and LRRK2 R1441C transgenic mice. Tyrosine hydroxylase (TH) staining (in green (shown as light grey in black and white image)) marks dopaminergic neurons.

FIG. 10B is a photographic image of an immunoblot analysis of N2a cells transiently expressing wildtype or mutant forms RAB7L1 as indicated. 30 μg of cell lysate was loaded in each lane. Arrrowheads indicate RAB7L1 as detected by an anti-GFP antibody; the DN form leads to a smaller product as expected.

FIG. 11 is a bar graph showing a negative geotaxis analysis of lrrk mutant Drosophila. Lrrk mutant (−/−) Drosophila defective negative geotaxis can be rescued by pan-neuronal expression of human LRRK2 WT or G2019S transgenes. Bars represent Mean times to climb 10 cm upward in a cylinder are represented. Error bars are SEM. ***: p<0.001, for comparison with the non-transgenic group by ANOVA (df=3, F=13.14) followed by Tukey HSD post hoc analysis.

FIG. 12A is a schematic showing the exons/introns structure of the RAB7L1 gene and its different known isoform products.

FIG. 12B is a schematic showing the exons/introns structure of an artificial RAB7L1 minigene and expected isoform products.

FIG. 12C is a schematic showing the RAB7L1 protein functional domains as predicted by CD-search, in parallel with the exonic structure of the CDS. The dashed red line indicates the alternative start site of the CDS in the event of exon 2 exclusion.

FIG. 12D is a photographic image of a gel showing rtPCR products to assess the splice of a RAB7L1 exon 2 reporter in SH-SY5Y cells transfected with a minigene bearing one of the two rs1572931 alleles. The numbered arrows correspond to the different isoforms expected from the minigene as depicted in FIG. 12B.

FIG. 12E is a graph showing the relative quantification of the different isoforms produced by the RAB7L1 minigene. The numbers correspond to the different isoforms expected from the minigene and shown in FIG. 12A.

FIG. 12F is a bar graph showing that alternatively spliced RAB7L1 (AT) does not functionally rescue LRRK2 G2019S neurite length assay. Total neurite length of primary rat cortical neurons transfected with expression vectors as indicated. (n=20 per group; ***p<0.001 vs vector alone by ANOVA followed by Bonferroni correction).

FIG. 13 is a photographic image of an immunoprecipitation using an anti-LRRK2 antibody from whole brain lysates of nontransgenic (NT), LRRK2 wild type transgenic (WT), LRRK2 R1441C (RC) transgenic, or LRRK2 knockout (−/−) mice. IB was subsequently performed for VPS35 and beta actin. VPS35 but not beta-actin were co-precipitated with LRRK2. Neither VPS35 nor beta-actin were immunoprecipitated by a control IgG antibody, or from LRRK2 KO mice.

FIG. 14 shows total neurite length per neuron in each tested condition. Black bars are vehicle-treated cells, grey bars are drug-treated cells. Neurons expressing vector alone, or VPS35 shRNA, VPS35 D620N, or LRRK2 G2019S were tested. ++ p<0.01; +++ p<0.001; ** p<0.01; *** p<0.001; n.s. no significant change. n=10-15 neurons per condition. Bars represent mean+SEM. p values calculated by ANOVA with Tukey's HSD post hoc.

DETAILED DESCRIPTION

The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.

Parkinson's disease (PD) is a common neurodegenerative disorder of aging, characterized by slowed movements and a distinctive tremor at rest (Lang and Lozano, 1998). Defining pathological features of the disease include neurodegeneration that is most prominent among midbrain dopamine neurons (DNs) in the Substantia Nigra (SN) and Lewy body protein aggregates that are composed in part of alpha-Synuclein (aSyn) protein. As the course of PD is thought to last decades, and as at the time of autopsy the vast majority of DNs are long lost, the molecular pursuit of initial etiological events has proven difficult.

In rare inherited familial forms of PD, specific causative mutations have been identified, and this has significantly advanced the field (Abeliovich and Beal, 2006; Hardy et al., 2006). For instance, autosomal dominantly inherited mutations in aSyn, including missense mutations and triplication of the locus, lead to a familial PD variant, implicating aSyn directly in the disease process. Another familial genetic cause of PD is the presence of autosomal dominantly inherited mutations in the Leucine rich-repeat kinase-2 (LRRK2) protein, which encodes a large multidomain protein with GTPase and kinase activities. Although the precise functions of aSyn and LRRK2 in neurons remain to be determined, both proteins have been broadly implicated in intraneuronal protein sorting. aSyn mutations have been reported to modify synaptic vesicle kinetics (Abeliovich et al., 2000) as well as trafficking to the Golgi apparatus in a variety of model systems (Cooper et al., 2006; Thayanidhi et al., 2010), whereas LRRK2 mutations are implicated in defective lysosomal protein degradation and macroautophagy, which is a cellular process that delivers cytosolic proteins and protein aggregates to the lysosome (Dodson et al., 2012; Heo et al., 2010; MacLeod et al., 2006), and Golgi Apparatus integrity (Stafa et al., 2012). The recent identification of rare autosomal dominant familial PD mutations in VPS35 (Vilarino-Guell et al., 2011; Zimprich et al., 2011), which encodes a component of the retromer complex that guides protein sorting from the endosome-lysosome degradation pathway retrogradely to the Golgi Apparatus (Bonifacino and Hurley, 2008; Seaman, 2009; Seaman et al., 1998), suggests that defective protein sorting in vesicular compartments may play a role in PD.

Several genome-wide association studies (GWAS) have described common genetic variants (at single nucleotide polymorphisms; SNPs) that modify PD risk in non-familial ‘sporadic’ cases (Hamza et al., 2010; Simon-Sanchez et al., 2009). Strikingly, a subset of these common variants lie within genomic loci previously associated with familial disease, such as aSyn or LRRK2, supporting the notion that common pathogenic pathways underlie familial and sporadic forms of PD. However, mechanisms that underlie the impact of non-familial genetic loci on PD risk, or that relate the functions of such loci to familial PD genes, remain unclear.

Described herein is a series of human brain transcriptome, human genetic, and cell biological studies, that together implicate a PD-associated genetic and cellular pathway. RAB7L1—one of 5 genes within the PARK16 non-familial PD risk-associated locus—functions together with LRRK2 to impact non-familial PD risk in the human population. This genetic interaction is apparent even in unaffected individuals who carry both risk alleles, as quantified in terms of a broad transcriptomic analysis of brain gene expression. Similarly, these genes together modify neuronal survival and neurite integrity in model systems. At a cellular level, defects in this PD-associated RAB7L1-LRRK2 pathway lead to abnormal lysosomal structures and defective retromer complex function, that normally links the endolysosomal protein degradation system with the Golgi apparatus (Bonifacino and Hurley, 2008; Seaman, 2009; Seaman et al., 1998). Consistent with a role for such cellular defects in disease pathology, mutations in a retromer complex component, VPS35, have recently been associated with rare forms of autosomal dominantly inherited familial PD (Vilarino-Guell et al., 2011; Zimprich et al., 2011).

Molecules of the Invention

As used herein, a “RAB7L1 molecule” refers to a RAB7L1 protein, or a fragment thereof. A “RAB7L1 molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to a RAB7L1 protein, or fragment thereof. For example, a RAB7L1 molecule can comprise the nucleic acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, or 5, or comprise the amino acid sequences shown in SEQ ID NOS: 6, 7, 8, or 26. For example, a RAB7L1 molecule can be encoded by a recombinant nucleic acid encoding a RAB7L1 protein, or fragment thereof. The RAB7L1 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a RAB7L1 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. A RAB7L1 molecule can include a fragment or portion of a RAB7L1 protein. A RAB7L1 molecule can include a variant of the above described examples, such as a fragment thereof. Such a variant can comprise a naturally-occurring variant due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms (e.g. SEQ ID NOS: 2-5). In one embodiment, a RAB7L1 molecule is encoded by a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NOS: 1, 2, 3, 4, or 5 wherein the variant has a nucleotide sequence identity to SEQ ID NOS: 1, 2, 3, 4, or 5 of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another embodiment, a variant of the RAB7L1 protein comprises a protein or polypeptide encoded by a RAB7L1 nucleic acid sequence, such as the sequence shown in SEQ ID NOS: 1, 2, 3, 4, or 5.

As used herein, a “LRRK2 molecule” refers to a LRRK2 protein, or a fragment thereof. A “LRRK2 molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to a LRRK2 protein, or fragment thereof. For example, a LRRK2 molecule can comprise the nucleic acid sequences shown in SEQ ID NOS: 9, or 10, or comprising the amino acid sequences shown in SEQ ID NO: 11, 27, or 28. For example, a LRRK2 molecule can be encoded by a recombinant nucleic acid encoding a LRRK2 protein, or fragment thereof. The LRRK2 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a LRRK2 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. A LRRK2 molecule can include a fragment or portion of a LRRK2 protein. A LRRK2 molecule can include a variant of the above described examples, such as a fragment thereof. Such a variant can comprise a naturally-occurring variant due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms. In one embodiment, a LRRK2 molecule is encoded by a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NOS: 9, or 10 wherein the variant has a nucleotide sequence identity to SEQ ID NOS: 9 or 10 of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another embodiment, a variant of the LRRK2 protein comprises a protein or polypeptide encoded by a LRRK2 nucleic acid sequence, such as the sequence shown in SEQ ID NOS: 9 or 10.

As used herein, a “VPS35 molecule” refers to a VPS35 protein, or a fragment thereof. A “VPS35 molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to a VPS35 protein, or fragment thereof. For example, a VPS35 molecule can comprise the nucleic acid sequences shown in SEQ ID NOS: 12 or 13, or comprising the amino acid sequences shown in SEQ ID NO: 14. For example, a VPS35 molecule can be encoded by a recombinant nucleic acid encoding a VPS35 protein, or fragment thereof. The VPS35 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a VPS35 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. A VPS35 molecule can include a fragment or portion of a VPS35 protein. A VPS35 molecule can include a variant of the above described examples, such as a fragment thereof. Such a variant can comprise a naturally-occurring variant due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms. In one embodiment, a VPS35 molecule is encoded by a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NOS: 12, or 13 wherein the variant has a nucleotide sequence identity to SEQ ID NOS: 12, or 13 of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another embodiment, a variant of the VPS35 protein comprises a protein or polypeptide encoded by a VPS35 nucleic acid sequence, such as the sequence shown in SEQ ID NOS: 12 or 13.

The nucleic acid can be any type of nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA. For example, a nucleic acid encoding a RAB7L1, a LRRK2, or a VPS35 protein can comprise a recombinant nucleic acid encoding such a protein. The nucleic acid can be a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences). It can be double-stranded or single-stranded.

The invention further provides for nucleic acids that are complementary to a RAB7L1, a LRRK2, or a VPS35 molecule. Complementary nucleic acids can hybridize to the nucleic acid sequence described above under stringent hybridization conditions. Non-limiting examples of stringent hybridization conditions include temperatures above 30° C., above 35° C., in excess of 42° C., and/or salinity of less than about 500 mM, or less than 200 mM. Hybridization conditions can be adjusted by the skilled artisan via modifying the temperature, salinity and/or the concentration of other reagents such as SDS or SSC.

According to the invention, protein variants can include amino acid sequence modifications. For example, amino acid sequence modifications fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions can include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. In one embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule can be modified with an amino acid sequence inserted as a carboxyl terminal fusion. For example, carboxyl terminal fusions may be used to increase the stability of a RAB7L1, a LRRK2, or a VPS35 molecule.

In one embodiment, a RAB7L1 molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding a RAB7L1 protein, such as the sequences shown in SEQ ID NOS: 6, 7, 8, or 26. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a RAB7L1 molecule is the polypeptide having the amino acid sequence shown in SEQ ID NOS: 6, 7, 8, or 26. Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NOS: 6, 7, 8, or 26 or having at least from about 50.1% to about 55% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 55.1% to about 60% identity to SEQ ID NOS: 6, 7, 8, or 26, or having from at least about 60.1% to about 65% identity to SEQ ID NOS: 6, 7, 8, or 26, or having from about 65.1% to about 70% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 70.1% to about 75% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 75.1% to about 80% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 80.1% to about 85% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 85.1% to about 90% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 90.1% to about 95% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 95.1% to about 97% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 97.1% to about 99% identity to SEQ ID NOS: 6, 7, 8, or 26. In another embodiment, a RAB7L1 molecule can be a fragment of a RAB7L1 protein.

In one embodiment, a LRRK2 molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding a LRRK2 protein, such as the sequences shown in SEQ ID NOS: 11, 27, or 28. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a LRRK2 molecule is the polypeptide having the amino acid sequence shown in SEQ ID NOS: 11, 27, or 28. Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NOS: 11, 27, or 28 or having at least from about 50.1% to about 55% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 55.1% to about 60% identity to SEQ ID NOS: 11, 27, or 28, or having from at least about 60.1% to about 65% identity to SEQ ID NOS: 11, 27, or 28, or having from about 65.1% to about 70% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 70.1% to about 75% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 75.1% to about 80% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 80.1% to about 85% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 85.1% to about 90% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 90.1% to about 95% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 95.1% to about 97% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 97.1% to about 99% identity to SEQ ID NOS: 11, 27, or 28. In another embodiment, a LRRK2 molecule can be a fragment of a LRRK2 protein.

In one embodiment, a VPS35 molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding a VPS35 protein, such as the sequences shown in SEQ ID NO: 14. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a VPS35 molecule is the polypeptide having the amino acid sequence shown in SEQ ID NO: 14. Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NO: 14 or having at least from about 50.1% to about 55% identity to SEQ ID NO: 14, or having at least from about 55.1% to about 60% identity to SEQ ID NO: 14, or having from at least about 60.1% to about 65% identity to SEQ ID NO: 14, or having from about 65.1% to about 70% identity to SEQ ID NO: 14, or having at least from about 70.1% to about 75% identity to SEQ ID NO: 14, or having at least from about 75.1% to about 80% identity to SEQ ID NO: 14, or having at least from about 80.1% to about 85% identity to SEQ ID NO: 14, or having at least from about 85.1% to about 90% identity to SEQ ID NO: 14, or having at least from about 90.1% to about 95% identity to SEQ ID NO: 14, or having at least from about 95.1% to about 97% identity to SEQ ID NO: 14, or having at least from about 97.1% to about 99% identity to SEQ ID NO: 14. In another embodiment, a VPS35 molecule can be a fragment of a VPS35 protein.

In one embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, according to the methods described herein can be administered to a subject as a recombinant protein. In another embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, can be administered to a subject as a modified recombinant protein. In a further embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, according to the methods described herein can be administered to a subject by delivery of a nucleic acid encoding a RAB7L1, a LRRK2, or a VPS35 protein, or fragment thereof. For example, nucleic acids can be delivered to a subject using a viral vector.

Polypeptides can be susceptible to denaturation or enzymatic degradation in the blood, liver or kidney. Accordingly, polypeptides can be unstable and have short biological half-lives. Polypeptides can be modified to increase their stability, for example, a fusion protein can be generated for increased stability and to cause a longer biological half-life to the polypeptides in circulation.

The term “biological half-life” is the time required for the activity of a substance taken into the body to lose one half its initial pharmacologic, physiologic, or biologic activity.

The invention provides for a nucleic acid encoding a RAB7L1 protein, or fragment thereof.

For example, the human genomic nucleotide sequence corresponding to the sense strand of the human RAB7L1 gene is depicted in SEQ ID NO: 1 (9752 bp). Sequence information related to RAB7L1 is accessible in public databases by GenBank Accession number NC_000001.10, 205737114, 205744615, complement (nucleotide).

SEQ ID NO: 1:    1 ctgaaagaaa aagataatca tcagagaagt acggggatga caaagaaaga acagcgtcat   61 agaaggcata agggaaacaa atgtcaagga gtggtcaact atgtcaaaac gaataagaac  121 agagaaaact ggatccttaa agatgagtag cttgaactaa cctcctgtac ctgggtaacg  181 aacattctgg gcaaaatata ttgtgaacca tgctgatgtt ctttcgccac aaaacataag  241 tgatagcctc tctatcaaga gacctagatt tcctagtgtc tggtcctggg ctgttttccc  301 atggccccgc tggttctctt cctctattcc tagaaaagta tagaaaatgg agctctcttt  361 gcttgccatt gagctcatgt cctgccaact tctacccaag tgacaggtag gtcagtaaag  421 ttacaggctg agaaattaaa attatttgcc aaacactagt tttatgatta tctattaaca  481 acagccacaa caaatcaaga aggagtccca gtgtatttca attagcttct agctcaagtg  541 atacatgagt agtatggcta gaataggcat tgtagaagta tgaatggaga actgtctctt  601 gttttctgtc catctttatt gttattgctg gatattaaat gctgtaattt ttggcatagg  661 tttttttcca aagactggtc tcttgggtca agttttcctg aacttgcctg gtattgggca  721 agttcacaca gaaaatatga gtcatttcac aaagtcctgg gcttgtctat ctacttgaag  781 gaataggaaa tggacttcac ttggagcatg atggatgagg cttgggcttt tactaatgaa  841 ataagtttca atcttaactc agttaaacct ttaaagacag gagaaaacta tctggggatg  901 gattcaggcc agtgagatca cgttgcacaa atctcccctt aagggatctt tactgaccta  961 gaattgtttc aaaataagat tccaaactgt cgagggagtt tgaagccttg aaacttggtg 1021 taggtttgct taaaacaaaa taacgaatat actacatttc ccaggaggct tcgcggtgag 1081 cctccgcact cggctggttc tctttaccgc gaggaaagct gggaaatgta gtgccacagg 1141 caaccctgca cgtgacgctt gcggaggaag gggagagaga ggcgcgcggg agggcgtcta 1201 gggaatcgag gtgccggctg ctccttcctc acaatttggt ttgtgctgca aggggagggt 1261 ccccatcatc tggccccagt ggtgtaagga gctgactggg attcagtcac tgacttggag 1321 ccgctcgggg gaagtcccgg tgggtgaggt tccgcggcgc ctggtccagt ttctcggcag 1381 tcaggccagg agggggtggg gaaggtgcga acagccgggg gctcggagct cgcgccctgc 1441 gccccaccca ggttgcggcc gcccggggag gaagcgcgta taggttgagt gcaaagtttc 1501 cttttttgct ttctcgtcag agcagcccag acaggcggtg ggtgggaatg cctcacttca 1561 gtttgaagag ggtccggatc caaaggggtt aaaacgagcg acccccgatc cccgaccaca 1621 cttcccgcct ccctaaaacg cacaccccgc tagccatggg cagccgcgac cacctgttca 1681 aagtgctggt ggtgggggac gccgcagtgg gcaagacgtc gctggtgcag cgatattccc 1741 aggacagctt cagcaaacac tacaagtcca cggtgggagg tgagacgtct cgggggtggg 1801 gagggggagg gggaggagga ggccccgacc gcggactcga gctggggacc caagtggggt 1861 ggggatttgc tgtcggttgt tgggtccttc ttcgggtcag gaagtactgt ctgggaacct 1921 agatggtgta attgccgggt gtttggttta cacgcctctc ttgagtttcc cgttagattt 1981 tcccaaactg agcccactgc caggagaaag gtaagagcaa aggcgtgaag atgagaaata 2041 gggaagggag gatgaggatg agcaattgct gtttattgca ggcgtactgt gaacttaagt 2101 ctccagcctt tcagctgtcc ggcaaatctc tccaatcagc agaagcagct gagatcctct 2161 gtggggcctc ctggttaggt gccggctggg gctgtagtcc tgcccctctg ttgagggagg 2221 aaccgcaagg tgtttaacct ggcttcagtg ggagaacgca ggaagcggaa ctggtctgaa 2281 ggactgtagc tttgttgtgg ggctgtgggt gggatggaag acacaaaatc cccgaacgag 2341 ccccttggct acgcagtcag atgcccatga gggctgatga gctgtgtttt tgtccgttga 2401 ttgcttaaat aaaggtaacc aaggaattct tccaagaatg tgaaatatac cacttctctg 2461 acctatcttc ccaccacttt tttcctcaaa ctggagaagg tagggaggaa ggggacaccg 2521 agttcattca tttctttccc gggtgtggcc tttggactca actatttttt cagaggcaga 2581 aatgccttat tgttaattag aaggtattta gctaggaaga gctggtgatg ggggagagta 2641 acttaaaatt ttatttctct ctgttaaaag aacttgtttc aaggggggaa aactcgtacc 2701 aatggaaaaa ttgaaaatgt gtgctcctgt tcatatggaa gtttaaaaag aaattaacaa 2761 acatttacat tgcaattaaa acattctcct taagaacccc cagtagggcc cagtaccgtg 2821 gttcatactt gcaatcccag cactttagga ggctgaggcg ggaggattgc ttgagcccag 2881 gagttcgaga ccagccatgg gaaacatggc aagaccccca cctctacaaa aattaaagaa 2941 gaaagtagcc gtggtcccag ctactcggga aactgagacg agaggattgc ttgaactggg 3001 gaggtcgagg cttcagtgag ccccgttcgc accactcact gcactgtagc ctgggcaaca 3061 gagtgagacc ctgtctcaaa aacaaaggaa caaaaaattt aagtctggca aaattaaagg 3121 ttcaaagttt cagacactct atcctgaccg ctctgccctc ccactcctaa ataatataag 3181 ggtccctatt tcacggattt gggttaaaag tggcctgcta ttaatagtta atttttaaca 3241 attgctgtag ttttccttta gcaaagaata taattggcta attttttttt cagtgtttag 3301 tctaaatttc ccttagcttt gattcctgtc acagatgtga aagataatat ttccaaacct 3361 cagaaattac tctgtttctc tccttagcca aaaggctttc cccatggaat acttactcag 3421 ccttaaggag tctcagcacc tgcagacttg gtggtagatt ccagcaggac tcttcagcaa 3481 catccacaca gtgtcttctt cttgggactc ctctgggacc ttttccattt attccattct 3541 tgcccaagat aatacctgca accttatggt ggatgtagag ttccccaggg tgtgttcttt 3601 tcataataca atttgattag aaaccaacag tgaaattgaa tgtggaagat acgaaagcaa 3661 ccacacttgt ctcatttccc ccttctcccc catcacccct tccctgagaa aaccccctaa 3721 atctcccaga aactatttaa cctgagaatg agaattgttt tcatagttac tcccaccccc 3781 agtcttggag acaaacagta gcaaataaat gagcttgaca gtagagtaaa agctggaaac 3841 ccctttggag agtgagtatg aatcatccat ctcacactaa atacacgcat gtggactcat 3901 ctcagtgaat tgagggctta aagttaaaca tatgggattg gagttgtgtg tccatagggt 3961 ttcactgcct atttgatttg agtttatccc tattaatttt ttacagtgaa attttattaa 4021 agtataatgt acatatattt tcagtggatt ttgctctgaa ggttctccag tggtctgact 4081 acgagatagt gcggcttcag ctgtgggata ttgcaggtaa agtgggaggg ctagatagga 4141 acaagggaag aaattttgat ttaactagaa agctctgagg aaaaaatggg gcagcaaagc 4201 cagaaattgt ttttctgaag ctggaatgat tgagctgctc tgaggacaca tgagctgaat 4261 aggtgcagag cttttgtaga tgttttgccc tgaatagatc tggagcagtc tctatgtgga 4321 tggaggggct ggcaaggtgg tggattgtcc agactatagg tggacactcc cacagcaggg 4381 gagggtgctt tttctaggtt taaggctgac cttggtgaaa aacaagatcc ccagaaaata 4441 gatacagtaa aaatagtgaa tccttactat gtgccaagca ttcttctaag tattttaaat 4501 tcactgactc attaatcctc acatcaaccc tatcataaaa gctactgctt ttggagaatt 4561 gcttgaacct gggaggtgga ggttgcagtg agcctagatg gcaccactgc actccagcct 4621 gggcaacagg agtgcaggaa actgtttcaa aaaaaaaaaa aaaaagcgac tgcttttatc 4681 ttcactttac aaatgagaaa tctgaagcac tgagagcttg atttgcccga agtcaaatag 4741 tatgcagcag agcagggatt tggactggag caatctgggt gcggaatctg ccttggaaat 4801 aattgggctt cattatccct ttatgggata gaaaaccaag agtcagaggg ggaaagtgag 4861 tggccccatc tgacacatat gcttactgcc ctcttctaca aggaatgggg agaggccggc 4921 tcttgggtca ccttgagaac tctccccttt ctccccaggg caggagcgct tcacctctat 4981 gacacgattg tattatcggg atgcctctgc ctgtgttatt atgtttgacg ttaccaatgc 5041 cactaccttc agcaacagcc agaggtggaa acaggaccta gacagcaagc tcacactacc 5101 caatggagag ccggtgccct gcctgctctt ggccaacaag gtatgtggcc aatctcagaa 5161 aatggtccct agcctctatc tgtggttaga aaggaagtaa aatgctctct gattctgggc 5221 atccatttcc ccttcttaag ttggcaaaat catctctacc tccttcaaag gatgttgagg 5281 gtgagtgaga ctttctttct tttttttttt tttgagacgg agtctcgctc tgttgctcag 5341 gctggagtgc agtggcgcga tctcggctca ctgccagctc tgcctcccag gttcacgcca 5401 ttctcttgcc tcagcctccc gagtagctgg gactacaggt gcccaccacc acacccagct 5461 aatttttttg tatttttagt agagactggg tttcattgtg ttagccagga tggtctcgct 5521 ctcctgacct catgatctgc ccgcctcagc ctcccaaagt gctgggatta caggcgtgag 5581 ccactgcgcc cagccaaggg tgagactttc tttgggaaga atgttgatta tagatttccg 5641 atgctggtgt tcttatccat tggtggaaat atgtttaaaa tccacagtat ctgagccata 5701 ttctttctta gttgcaggaa gtttatgata ctgcctttta ttttttctct ttgctagtgt 5761 gatctgtccc cttgggcagt gagccgggac cagattgacc ggttcagtaa agagaacggt 5821 ttcacaggtt ggacagaaac atcagtcaag gagaacaaaa atattaatga ggctatgagg 5881 taagtagctc atgctgatag gcatgcagat gtatccatct tccactgtgg ccctgtggac 5941 cctcttttct tatttctgag ccaacaatga cagactgagc cattggaatg ctagccccta 6001 aaggtcaggg atactgtctt tcctgagata actggggaca aaggggcatt taaaccttct 6061 ggctttacca aatactctct gatgataagt cagactacat ctgccatttc tgcttttcct 6121 ccaaaataag cttctcaggc ttattttttg tcttttagag tcctcattga aaagatgatg 6181 agaaattcca cagaagatat catgtctttg tccacccaag gggactacat caatctacaa 6241 accaagtcct ccagctggtc ctgctgctag tagtgtttgg cttattttcc atcccagttc 6301 tgggaggtct tttaagtctc ttccctttgg ttgcccacct gacaatttta ttaagtacat 6361 ttgaattgtc tcctgactac tgtccagtaa ggaggcccat tgtcacttag aaaagacacc 6421 tggaacccat gtgcatttct gcatctcctg gattagcctt tcacatgttg ctggctcaca 6481 ttagtgccag ttagtgcctt cggtgtaaga tcttctcatc agccctcaat ttgtgatccg 6541 gaattttatg agaaggatta gaaatcagca cctgcgtttt agagatcata attctcacct 6601 acttctgagc ttatttttcc atttgatatt cattgatatc atgacttcca attgagagga 6661 aaatgagatc aaatgtcatt tcccaaattt cttgtaggcc gttgtttcag attctttctg 6721 tcttggaatg taaacatctg attctggaat gcagaaggag ggggtctggg catctgtgga 6781 tttttggcta ctagaagtgt cccagaagtc actgtatttt tgaaacttct aacgtcataa 6841 ttaagtttct cttgtcttgg catcaagaat agtcaagttt tttggccggg catggtggct 6901 catgcctgta atcccagcac ttggggaggc caaggcaggc ggatcacatg aggccaggaa 6961 ttcgagacca acctggtcag catggcaaaa ccccgtctct actaaaagta caaaaattag 7021 ccaggcgtga tggcacgtgt ctgtaatccc agctactctg gagactgagg tgggagaatc 7081 gcttgagact gggaggcaga ggttgcagtg aaccgagatc atgccaccgc acttcagcct 7141 gggtgacaga gaaggactcc gtctcaaaaa aaaaagaaaa aagaatagtc atttttaaac 7201 tacctatctc atgcaatgaa agcattttct tccacaaaga gcttaatcct catgatagga 7261 ttgcctagtg tctcccattt gcaggtttct gggttgatgt cttaatgcat aatactgcaa 7321 gtgacatcag ctggctgtga tgcttcgaaa taggtctgct cctcacagct ttgggaatct 7381 gaatggaaga agaaaagaga gaagttaaca acctccactg gggcaacttt gtgaacacgt 7441 aggcacttag tcataggaaa catattatgt gcaggtccta gcctggggga ggaaagtaga 7501 tagacagaaa atcattaggt aatttaagta ctaaattggg cagggctttt tagtatcaaa 7561 tcactactag accatttaat ttgttaaatt atctctagga tggtgattta taacctaccc 7621 aaagttatcg atattcttac taaactctga ggcctgaagt tctgtgatag accttaaata 7681 agtgtcctaa gtcagtggtt cccaaatctg gctggtcggg aatacctggg aagtttgtta 7741 aaatttttta aaaatgtttt aagatttttg ggtcctgagc cagccgtggt ggctcacacc 7801 tgtaatccca gcactttggg aggctgaggc aggtggatcg cctgaggtca ggagttcaag 7861 atcaacctgg ccaacatact gaaaccccgt ctctactaaa aataagaaaa attagctggg 7921 cgtggtggcg ggcacctgta atcccagcta cttgggagac tgaggcagga gaatcgcttg 7981 aacctgggag ttagaggttg cagtgagctg agatcacacc attgcgcttc agcctgggca 8041 acaagagtga aactccatct ccaaaaaaaa aaaacaaaaa gaaaaagatt tttgggtccc 8101 gacctcaaac ctactgaatc agaatttcta gggatgaagc ctaggaatgt gttgttgttt 8161 tcagagcttc cctggtgatt gtgataagcc tggtttggaa accattgctg gagaactttg 8221 taaagataca gagacccaga ccttttgtat ttacatttaa atacaaatac aaatcctggg 8281 tttctatata ttctgttagc ttttcaggtg attctgctac acagacgttg aaaaccactg 8341 ccctaagaaa gagatcagag gccacatatc agagagaaaa gggaccaaac cttcggtggt 8401 ttgttgtgtg tcgttttaat gccaattatt ttaacttgca cagtcttctg aaaccttgta 8461 ttaatagttc tcttttgtat taccattttc aggtagggtt ttgatcacta tgattctgaa 8521 gataatagtg aaatagtgaa tttcattgat atgaagagat aattgatttt cattcattgg 8581 tttgaacacc tgcaaaatca caaataaatg agaactaagt cttgtattca tggtggttat 8641 tggcctttaa tgtgagtttg tcaaagtgct gttttatact gatagctcaa gaggattgcg 8701 gaaatggaga ctttattttt taaatacctt tttctaaatt ttcaattcaa ggggtacgtg 8761 tgcaagtttg ttacatggat atattgtgtg atgctaaagc ttgggtttct gcttagcctg 8821 tctcccaagt agtgaacata gcacccagtc ggcacttttt caacgcttac tccctctctc 8881 cccacttttg ggagtctcca gtgtctgttg ttcccatctt tatgtgtgtg cccaatgttt 8941 acctcccact tacaagtgag aaatgtggtt aaataccttc ttttaaaatg aaggtaaata 9001 ttattctgtt tacctgttaa cttactaata ccaattgctt agatccatct gcctagtacc 9061 aattcagtac ttgatggaac atgccaaata ctacttggaa acgtgacctt tttgaaaaac 9121 tgaatgtgct tgcttttccc atttgctgac tcaggattta cctgcctttt caagcattgt 9181 tagaggctgg ccagccagcc agcccttctg gatattctct cccaactggt gaacattata 9241 ggcctgaaac taattaaaga aggaactgaa atatacctaa ttcctcttcg tgcccctatc 9301 cataagagaa aagcaattat gattaggaaa aataaaatat ttacatgaat ggaaatgcga 9361 tgctactaaa aataattagg tcttatcact cggatgctac ctctttggtg cattcaaaag 9421 aggtagcgtc caaaattgtt tggaacgtag atatcgtagc ccctccctag aggaggtcct 9481 acatttgaga caagatgcct ctccaaccac tcaggtggct tttggtcaaa aaagaaaaca 9541 gttcccccag agtgttctca gaaaccctaa gggagggctg agaacacagc tgtggggtag 9601 tgtctgccct gactttcttc tttgaacaga agaaatttgg tcacttagct gagtgaccaa 9661 ataagtggag cagaggagat tgtaggtcag ggaaggccct ttagcagtaa agaatggtct 9721 gctgctttat cactactatt gcgccagaat ta

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 1) is depicted in SEQ ID NO: 2 (3324 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1238, 1526-1779, 4045-4116, 4959-5140, 5758-5879, and 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 1). Sequence information related to RAB7L1 (transcript variant 1) is accessible in public databases by GenBank Accession number NM 003929.2 (nucleotide).

SEQ ID NO: 2:    1 agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg   61 gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgg tttgtgctgc  121 aaggggaggg tccccatcat ctggccccag tggtgtaagg agctgactgg gattcagtca  181 ctgacttgga gccgctcggg ggaagtcccg cccagacagg cggtgggtgg gaatgcctca  241 cttcagtttg aagagggtcc ggatccaaag gggttaaaac gagcgacccc cgatccccga  301 ccacacttcc cgcctcccta aaacgcacac cccgctagcc  atg ggcagcc gcgaccacct  361 gttcaaagtg ctggtggtgg gggacgccgc agtgggcaag acgtcgctgg tgcagcgata  421 ttcccaggac agcttcagca aacactacaa gtccacggtg ggagtggatt ttgctctgaa  481 ggttctccag tggtctgact acgagatagt gcggcttcag ctgtgggata ttgcagggca  541 ggagcgcttc acctctatga cacgattgta ttatcgggat gcctctgcct gtgttattat  601 gtttgacgtt accaatgcca ctaccttcag caacagccag aggtggaaac aggacctaga  661 cagcaagctc acactaccca atggagagcc ggtgccctgc ctgctcttgg ccaacaagtg  721 tgatctgtcc ccttgggcag tgagccggga ccagattgac cggttcagta aagagaacgg  781 tttcacaggt tggacagaaa catcagtcaa ggagaacaaa aatattaatg aggctatgag  841 agtcctcatt gaaaagatga tgagaaattc cacagaagat atcatgtctt tgtccaccca  901 aggggactac atcaatctac aaaccaagtc ctccagctgg tcctgctgct agtagtgttt  961 ggcttatttt ccatcccagt tctgggaggt cttttaagtc tcttcccttt ggttgcccac 1021 ctgacaattt tattaagtac atttgaattg tctcctgact actgtccagt aaggaggccc 1081 attgtcactt agaaaagaca cctggaaccc atgtgcattt ctgcatctcc tggattagcc 1141 tttcacatgt tgctggctca cattagtgcc agttagtgcc ttcggtgtaa gatcttctca 1201 tcagccctca atttgtgatc cggaatttta tgagaaggat tagaaatcag cacctgcgtt 1261 ttagagatca taattctcac ctacttctga gcttattttt ccatttgata ttcattgata 1321 tcatgacttc caattgagag gaaaatgaga tcaaatgtca tttcccaaat ttcttgtagg 1381 ccgttgtttc agattctttc tgtcttggaa tgtaaacatc tgattctgga atgcagaagg 1441 agggggtctg ggcatctgtg gatttttggc tactagaagt gtcccagaag tcactgtatt 1501 tttgaaactt ctaacgtcat aattaagttt ctcttgtctt ggcatcaaga atagtcaagt 1561 tttttggccg ggcatggtgg ctcatgcctg taatcccagc acttggggag gccaaggcag 1621 gcggatcaca tgaggccagg aattcgagac caacctggtc agcatggcaa aaccccgtct 1681 ctactaaaag tacaaaaatt agccaggcgt gatggcacgt gtctgtaatc ccagctactc 1741 tggagactga ggtgggagaa tcgcttgaga ctgggaggca gaggttgcag tgaaccgaga 1801 tcatgccacc gcacttcagc ctgggtgaca gagaaggact ccgtctcaaa aaaaaaagaa 1861 aaaagaatag tcatttttaa actacctatc tcatgcaatg aaagcatttt cttccacaaa 1921 gagcttaatc ctcatgatag gattgcctag tgtctcccat ttgcaggttt ctgggttgat 1981 gtcttaatgc ataatactgc aagtgacatc agctggctgt gatgcttcga aataggtctg 2041 ctcctcacag ctttgggaat ctgaatggaa gaagaaaaga gagaagttaa caacctccac 2101 tggggcaact ttgtgaacac gtaggcactt agtcatagga aacatattat gtgcaggtcc 2161 tagcctgggg gaggaaagta gatagacaga aaatcattag gtaatttaag tactaaattg 2221 ggcagggctt tttagtatca aatcactact agaccattta atttgttaaa ttatctctag 2281 gatggtgatt tataacctac ccaaagttat cgatattctt actaaactct gaggcctgaa 2341 gttctgtgat agaccttaaa taagtgtcct aagtcagtgg ttcccaaatc tggctggtcg 2401 ggaatacctg ggaagtttgt taaaattttt taaaaatgtt ttaagatttt tgggtcctga 2461 gccagccgtg gtggctcaca cctgtaatcc cagcactttg ggaggctgag gcaggtggat 2521 cgcctgaggt caggagttca agatcaacct ggccaacata ctgaaacccc gtctctacta 2581 aaaataagaa aaattagctg ggcgtggtgg cgggcacctg taatcccagc tacttgggag 2641 actgaggcag gagaatcgct tgaacctggg agttagaggt tgcagtgagc tgagatcaca 2701 ccattgcgct tcagcctggg caacaagagt gaaactccat ctccaaaaaa aaaaaacaaa 2761 aagaaaaaga tttttgggtc ccgacctcaa acctactgaa tcagaatttc tagggatgaa 2821 gcctaggaat gtgttgttgt tttcagagct tccctggtga ttgtgataag cctggtttgg 2881 aaaccattgc tggagaactt tgtaaagata cagagaccca gaccttttgt atttacattt 2941 aaatacaaat acaaatcctg ggtttctata tattctgtta gcttttcagg tgattctgct 3001 acacagacgt tgaaaaccac tgccctaaga aagagatcag aggccacata tcagagagaa 3061 aagggaccaa accttcggtg gtttgttgtg tgtcgtttta atgccaatta ttttaacttg 3121 cacagtcttc tgaaaccttg tattaatagt tctcttttgt attaccattt tcaggtaggg 3181 ttttgatcac tatgattctg aagataatag tgaaatagtg aatttcattg atatgaagag 3241 ataattgatt ttcattcatt ggtttgaaca cctgcaaaat cacaaataaa tgagaactaa 3301 gtcttgtaaa aaaaaaaaaa aaaa

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 2) is depicted in SEQ ID NO: 3 (3223 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1238, 1526-1779, 4045-4116, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 2). Sequence information related to RAB7L1 (transcript variant 2) is accessible in public databases by GenBank Accession number NM_001135662.1 (nucleotide).

SEQ ID NO: 3:    1 agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg   61 gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgc ccagacaggc  121 ggtgggtggg aatgcctcac ttcagtttga agagggtccg gatccaaagg ggttaaaacg  181 agcgaccccc gatccccgac cacacttccc gcctccctaa aacgcacacc ccgctagcc a  241 tg ggcagccg cgaccacctg ttcaaagtgc tggtggtggg ggacgccgca gtgggcaaga  301 cgtcgctggt gcagcgatat tcccaggaca gcttcagcaa acactacaag tccacggtgg  361 gagtggattt tgctctgaag gttctccagt ggtctgacta cgagatagtg cggcttcagc  421 tgtgggatat tgcagggcag gagcgcttca cctctatgac acgattgtat tatcgggatg  481 cctctgcctg tgttattatg tttgacgtta ccaatgccac taccttcagc aacagccaga  541 ggtggaaaca ggacctagac agcaagctca cactacccaa tggagagccg gtgccctgcc  601 tgctcttggc caacaagtgt gatctgtccc cttgggcagt gagccgggac cagattgacc  661 ggttcagtaa agagaacggt ttcacaggtt ggacagaaac atcagtcaag gagaacaaaa  721 atattaatga ggctatgaga gtcctcattg aaaagatgat gagaaattcc acagaagata  781 tcatgtcttt gtccacccaa ggggactaca tcaatctaca aaccaagtcc tccagctggt  841 cctgctgcta gtagtgtttg gcttattttc catcccagtt ctgggaggtc ttttaagtct  901 cttccctttg gttgcccacc tgacaatttt attaagtaca tttgaattgt ctcctgacta  961 ctgtccagta aggaggccca ttgtcactta gaaaagacac ctggaaccca tgtgcatttc 1021 tgcatctcct ggattagcct ttcacatgtt gctggctcac attagtgcca gttagtgcct 1081 tcggtgtaag atcttctcat cagccctcaa tttgtgatcc ggaattttat gagaaggatt 1141 agaaatcagc acctgcgttt tagagatcat aattctcacc tacttctgag cttatttttc 1201 catttgatat tcattgatat catgacttcc aattgagagg aaaatgagat caaatgtcat 1261 ttcccaaatt tcttgtaggc cgttgtttca gattctttct gtcttggaat gtaaacatct 1321 gattctggaa tgcagaagga gggggtctgg gcatctgtgg atttttggct actagaagtg 1381 tcccagaagt cactgtattt ttgaaacttc taacgtcata attaagtttc tcttgtcttg 1441 gcatcaagaa tagtcaagtt ttttggccgg gcatggtggc tcatgcctgt aatcccagca 1501 cttggggagg ccaaggcagg cggatcacat gaggccagga attcgagacc aacctggtca 1561 gcatggcaaa accccgtctc tactaaaagt acaaaaatta gccaggcgtg atggcacgtg 1621 tctgtaatcc cagctactct ggagactgag gtgggagaat cgcttgagac tgggaggcag 1681 aggttgcagt gaaccgagat catgccaccg cacttcagcc tgggtgacag agaaggactc 1741 cgtctcaaaa aaaaaagaaa aaagaatagt catttttaaa ctacctatct catgcaatga 1801 aagcattttc ttccacaaag agcttaatcc tcatgatagg attgcctagt gtctcccatt 1861 tgcaggtttc tgggttgatg tcttaatgca taatactgca agtgacatca gctggctgtg 1921 atgcttcgaa ataggtctgc tcctcacagc tttgggaatc tgaatggaag aagaaaagag 1981 agaagttaac aacctccact ggggcaactt tgtgaacacg taggcactta gtcataggaa 2041 acatattatg tgcaggtcct agcctggggg aggaaagtag atagacagaa aatcattagg 2101 taatttaagt actaaattgg gcagggcttt ttagtatcaa atcactacta gaccatttaa 2161 tttgttaaat tatctctagg atggtgattt ataacctacc caaagttatc gatattctta 2221 ctaaactctg aggcctgaag ttctgtgata gaccttaaat aagtgtccta agtcagtggt 2281 tcccaaatct ggctggtcgg gaatacctgg gaagtttgtt aaaatttttt aaaaatgttt 2341 taagattttt gggtcctgag ccagccgtgg tggctcacac ctgtaatccc agcactttgg 2401 gaggctgagg caggtggatc gcctgaggtc aggagttcaa gatcaacctg gccaacatac 2461 tgaaaccccg tctctactaa aaataagaaa aattagctgg gcgtggtggc gggcacctgt 2521 aatcccagct acttgggaga ctgaggcagg agaatcgctt gaacctggga gttagaggtt 2581 gcagtgagct gagatcacac cattgcgctt cagcctgggc aacaagagtg aaactccatc 2641 tccaaaaaaa aaaaacaaaa agaaaaagat ttttgggtcc cgacctcaaa cctactgaat 2701 cagaatttct agggatgaag cctaggaatg tgttgttgtt ttcagagctt ccctggtgat 2761 tgtgataagc ctggtttgga aaccattgct ggagaacttt gtaaagatac agagacccag 2821 accttttgta tttacattta aatacaaata caaatcctgg gtttctatat attctgttag 2881 cttttcaggt gattctgcta cacagacgtt gaaaaccact gccctaagaa agagatcaga 2941 ggccacatat cagagagaaa agggaccaaa ccttcggtgg tttgttgtgt gtcgttttaa 3001 tgccaattat tttaacttgc acagtcttct gaaaccttgt attaatagtt ctcttttgta 3061 ttaccatttt caggtagggt tttgatcact atgattctga agataatagt gaaatagtga 3121 atttcattga tatgaagaga taattgattt tcattcattg gtttgaacac ctgcaaaatc 3181 acaaataaat gagaactaag tcttgtaaaa aaaaaaaaaa aaa

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 3) is depicted in SEQ ID NO: 4 (3438 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1779, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 3). Sequence information related to RAB7L1 (transcript variant 3) is accessible in public databases by GenBank Accession number NM_001135663.1 (nucleotide).

SEQ ID NO: 4:    1 agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg   61 gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgg tttgtgctgc  121 aaggggaggg tccccatcat ctggccccag tggtgtaagg agctgactgg gattcagtca  181 ctgacttgga gccgctcggg ggaagtcccg gtgggtgagg ttccgcggcg cctggtccag  241 tttctcggca gtcaggccag gagggggtgg ggaaggtgcg aacagccggg ggctcggagc  301 tcgcgccctg cgccccaccc aggttgcggc cgcccgggga ggaagcgcgt ataggttgag  361 tgcaaagttt ccttttttgc tttctcgtca gagcagccca gacaggcggt gggtgggaat  421 gcctcacttc agtttgaaga gggtccggat ccaaaggggt taaaacgagc gacccccgat  481 ccccgaccac acttcccgcc tccctaaaac gcacaccccg ctagcc atg g gcagccgcga  541 ccacctgttc aaagtgctgg tggtggggga cgccgcagtg ggcaagacgt cgctggtgca  601 gcgatattcc caggacagct tcagcaaaca ctacaagtcc acggtgggag ggcaggagcg  661 cttcacctct atgacacgat tgtattatcg ggatgcctct gcctgtgtta ttatgtttga  721 cgttaccaat gccactacct tcagcaacag ccagaggtgg aaacaggacc tagacagcaa  781 gctcacacta cccaatggag agccggtgcc ctgcctgctc ttggccaaca agtgtgatct  841 gtccccttgg gcagtgagcc gggaccagat tgaccggttc agtaaagaga acggtttcac  901 aggttggaca gaaacatcag tcaaggagaa caaaaatatt aatgaggcta tgagagtcct  961 cattgaaaag atgatgagaa attccacaga agatatcatg tctttgtcca cccaagggga 1021 ctacatcaat ctacaaacca agtcctccag ctggtcctgc tgctagtagt gtttggctta 1081 ttttccatcc cagttctggg aggtctttta agtctcttcc ctttggttgc ccacctgaca 1141 attttattaa gtacatttga attgtctcct gactactgtc cagtaaggag gcccattgtc 1201 acttagaaaa gacacctgga acccatgtgc atttctgcat ctcctggatt agcctttcac 1261 atgttgctgg ctcacattag tgccagttag tgccttcggt gtaagatctt ctcatcagcc 1321 ctcaatttgt gatccggaat tttatgagaa ggattagaaa tcagcacctg cgttttagag 1381 atcataattc tcacctactt ctgagcttat ttttccattt gatattcatt gatatcatga 1441 cttccaattg agaggaaaat gagatcaaat gtcatttccc aaatttcttg taggccgttg 1501 tttcagattc tttctgtctt ggaatgtaaa catctgattc tggaatgcag aaggaggggg 1561 tctgggcatc tgtggatttt tggctactag aagtgtccca gaagtcactg tatttttgaa 1621 acttctaacg tcataattaa gtttctcttg tcttggcatc aagaatagtc aagttttttg 1681 gccgggcatg gtggctcatg cctgtaatcc cagcacttgg ggaggccaag gcaggcggat 1741 cacatgaggc caggaattcg agaccaacct ggtcagcatg gcaaaacccc gtctctacta 1801 aaagtacaaa aattagccag gcgtgatggc acgtgtctgt aatcccagct actctggaga 1861 ctgaggtggg agaatcgctt gagactggga ggcagaggtt gcagtgaacc gagatcatgc 1921 caccgcactt cagcctgggt gacagagaag gactccgtct caaaaaaaaa agaaaaaaga 1981 atagtcattt ttaaactacc tatctcatgc aatgaaagca ttttcttcca caaagagctt 2041 aatcctcatg ataggattgc ctagtgtctc ccatttgcag gtttctgggt tgatgtctta 2101 atgcataata ctgcaagtga catcagctgg ctgtgatgct tcgaaatagg tctgctcctc 2161 acagctttgg gaatctgaat ggaagaagaa aagagagaag ttaacaacct ccactggggc 2221 aactttgtga acacgtaggc acttagtcat aggaaacata ttatgtgcag gtcctagcct 2281 gggggaggaa agtagataga cagaaaatca ttaggtaatt taagtactaa attgggcagg 2341 gctttttagt atcaaatcac tactagacca tttaatttgt taaattatct ctaggatggt 2401 gatttataac ctacccaaag ttatcgatat tcttactaaa ctctgaggcc tgaagttctg 2461 tgatagacct taaataagtg tcctaagtca gtggttccca aatctggctg gtcgggaata 2521 cctgggaagt ttgttaaaat tttttaaaaa tgttttaaga tttttgggtc ctgagccagc 2581 cgtggtggct cacacctgta atcccagcac tttgggaggc tgaggcaggt ggatcgcctg 2641 aggtcaggag ttcaagatca acctggccaa catactgaaa ccccgtctct actaaaaata 2701 agaaaaatta gctgggcgtg gtggcgggca cctgtaatcc cagctacttg ggagactgag 2761 gcaggagaat cgcttgaacc tgggagttag aggttgcagt gagctgagat cacaccattg 2821 cgcttcagcc tgggcaacaa gagtgaaact ccatctccaa aaaaaaaaaa caaaaagaaa 2881 aagatttttg ggtcccgacc tcaaacctac tgaatcagaa tttctaggga tgaagcctag 2941 gaatgtgttg ttgttttcag agcttccctg gtgattgtga taagcctggt ttggaaacca 3001 ttgctggaga actttgtaaa gatacagaga cccagacctt ttgtatttac atttaaatac 3061 aaatacaaat cctgggtttc tatatattct gttagctttt caggtgattc tgctacacag 3121 acgttgaaaa ccactgccct aagaaagaga tcagaggcca catatcagag agaaaaggga 3181 ccaaaccttc ggtggtttgt tgtgtgtcgt tttaatgcca attattttaa cttgcacagt 3241 cttctgaaac cttgtattaa tagttctctt ttgtattacca ttttcaggt agggttttga 3301 tcactatgat tctgaagata atagtgaaat agtgaatttca ttgatatga agagataatt 3361 gattttcatt cattggtttg aacacctgca aaatcacaaat aaatgagaa ctaagtcttg 3421 taaaaaaaaa aaaaaaaa

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 4) is depicted in SEQ ID NO: 5 (3070 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1339, 4045-4116, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 4). Sequence information related to RAB7L1 (transcript variant 4) is accessible in public databases by GenBank Accession number NM_001135664.1 (nucleotide).

SEQ ID NO: 5:    1 agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg   61 gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgg tttgtgctgc  121 aaggggaggg tccccatcat ctggccccag tggtgtaagg agctgactgg gattcagtca  181 ctgacttgga gccgctcggg ggaagtcccg tggattttgc tctgaaggtt ctccagtggt  241 ctgactacga gatagtgcgg cttcagctgt gggatattgc agggcaggag cgcttcacct  301 ct atg acacg attgtattat cgggatgcct ctgcctgtgt tattatgttt gacgttacca  361 atgccactac cttcagcaac agccagaggt ggaaacagga cctagacagc aagctcacac  421 tacccaatgg agagccggtg ccctgcctgc tcttggccaa caagtgtgat ctgtcccctt  481 gggcagtgag ccgggaccag attgaccggt tcagtaaaga gaacggtttc acaggttgga  541 cagaaacatc agtcaaggag aacaaaaata ttaatgaggc tatgagagtc ctcattgaaa  601 agatgatgag aaattccaca gaagatatca tgtctttgtc cacccaaggg gactacatca  661 atctacaaac caagtcctcc agctggtcct gctgctagta gtgtttggct tattttccat  721 cccagttctg ggaggtcttt taagtctctt ccctttggtt gcccacctga caattttatt  781 aagtacattt gaattgtctc ctgactactg tccagtaagg aggcccattg tcacttagaa  841 aagacacctg gaacccatgt gcatttctgc atctcctgga ttagcctttc acatgttgct  901 ggctcacatt agtgccagtt agtgccttcg gtgtaagatc ttctcatcag ccctcaattt  961 gtgatccgga attttatgag aaggattaga aatcagcacc tgcgttttag agatcataat 1021 tctcacctac ttctgagctt atttttccat ttgatattca ttgatatcat gacttccaat 1081 tgagaggaaa atgagatcaa atgtcatttc ccaaatttct tgtaggccgt tgtttcagat 1141 tctttctgtc ttggaatgta aacatctgat tctggaatgc agaaggaggg ggtctgggca 1201 tctgtggatt tttggctact agaagtgtcc cagaagtcac tgtatttttg aaacttctaa 1261 cgtcataatt aagtttctct tgtcttggca tcaagaatag tcaagttttt tggccgggca 1321 tggtggctca tgcctgtaat cccagcactt ggggaggcca aggcaggcgg atcacatgag 1381 gccaggaatt cgagaccaac ctggtcagca tggcaaaacc ccgtctctac taaaagtaca 1441 aaaattagcc aggcgtgatg gcacgtgtct gtaatcccag ctactctgga gactgaggtg 1501 ggagaatcgc ttgagactgg gaggcagagg ttgcagtgaa ccgagatcat gccaccgcac 1561 ttcagcctgg gtgacagaga aggactccgt ctcaaaaaaa aaagaaaaaa gaatagtcat 1621 ttttaaacta cctatctcat gcaatgaaag cattttcttc cacaaagagc ttaatcctca 1681 tgataggatt gcctagtgtc tcccatttgc aggtttctgg gttgatgtct taatgcataa 1741 tactgcaagt gacatcagct ggctgtgatg cttcgaaata ggtctgctcct cacagcttt 1801 gggaatctga atggaagaag aaaagagaga agttaacaac ctccactgggg caactttgt 1861 gaacacgtag gcacttagtc ataggaaaca tattatgtgc aggtcctagcc tgggggagg 1921 aaagtagata gacagaaaat cattaggtaa tttaagtact aaattgggcag ggcttttta 1981 gtatcaaatc actactagac catttaattt gttaaattat ctctaggatgg tgatttata 2041 acctacccaa agttatcgat attcttacta aactctgagg cctgaagttct gtgatagac 2101 cttaaataag tgtcctaagt cagtggttcc caaatctggc tggtcgggaat acctgggaa 2161 gtttgttaaa attttttaaa aatgttttaa gatttttggg tcctgagccag ccgtggtgg 2221 ctcacacctg taatcccagc actttgggag gctgaggcag gtggatcgcct gaggtcagg 2281 agttcaagat caacctggcc aacatactga aaccccgtct ctactaaaaat aagaaaaat 2341 tagctgggcg tggtggcggg cacctgtaat cccagctact tgggagactga ggcaggaga 2401 atcgcttgaa cctgggagtt agaggttgca gtgagctgag atcacaccatt gcgcttcag 2461 cctgggcaac aagagtgaaa ctccatctcc aaaaaaaaaa aacaaaaagaa aaagatttt 2521 tgggtcccga cctcaaacct actgaatcag aatttctagg gatgaagccta ggaatgtgt 2581 tgttgttttc agagcttccc tggtgattgt gataagcctg gtttggaaacc attgctgga 2641 gaactttgta aagatacaga gacccagacc ttttgtattt acatttaaata caaatacaa 2701 atcctgggtt tctatatatt ctgttagctt ttcaggtgat tctgctacaca gacgttgaa 2761 aaccactgcc ctaagaaaga gatcagaggc cacatatcag agagaaaaggg accaaacct 2821 tcggtggttt gttgtgtgtc gttttaatgc caattatttt aacttgcacag tcttctgaa 2881 accttgtatt aatagttctc ttttgtatta ccattttcag gtagggttttg atcactatg 2941 attctgaaga taatagtgaa atagtgaatt tcattgatat gaagagataat tgattttca 3001 ttcattggtt tgaacacctg caaaatcaca aataaatgag aactaagtctt gtaaaaaaa 3061 aaaaaaaaaa

For example, other mRNA transcript variants of human RAB7L1 can exist. For example nucleotides 1130-1238, 4045-4116, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 5). Sequence information related to RAB7L1 transcript variants is accessible in public databases such as GenBank.

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 1) is encoded by the nucleic acid sequence of SEQ ID NOS: 2 or 3 and is depicted in SEQ ID NO: 6 (203aa). Sequence information related to RAB7L1 (isoform 1) is accessible in public databases by GenBank Accession numbers NP_003920.1 and NP_001129134.1 (protein).

SEQ ID NO: 6:   1 MGSRDHLFKV LVVGDAAVGK TSLVQRYSQD SFSKHYKSTV GVDFALKVLQ WSDYEIVRLQ  61 LWDIAGQERF TSMTRLYYRD ASACVIMFDV TNATTFSNSQ RWKQDLDSKL TLPNGEPVPC 121 LLLANKCDLS PWAVSRDQID RFSKENGFTG WTETSVKENK NINEAMRVLI EKMMRNSTED 181 IMSLSTQGDY INLQTKSSSW SCC

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 2) is encoded by the nucleic acid sequence of SEQ ID NO: 4 and is depicted in SEQ ID NO: 7 (179aa). Sequence information related to RAB7L1 (isoform 2) is accessible in public databases by GenBank Accession numbers NP_001123135.1 (protein).

SEQ ID NO: 7:   1 MGSRDHLFKV LVVGDAAVGK TSLVQRYSQD SFSKHYKSTV GGQERFTSMT RLYYRDASAC  61 VIMFDVTNAT TFSNSQRWKQ DLDSKLTLPN GEPVPCLLLA NKCDLSPWAV SRDQIDRFSK 121 ENGFTGWTET SVKENKNINE AMRVLIEKMM RNSTEDIMSL STQGDYINLQ TKSSSWSCC

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 3) is encoded by the nucleic acid sequence of SEQ ID NO: 5 and is depicted in SEQ ID NO: 8 (131aa). Sequence information related to RAB7L1 (isoform 3) is accessible in public databases by GenBank Accession numbers NP_001129136.1 (protein).

SEQ ID NO: 8:   1 MTRLYYRDAS ACVIMFDVTN ATTFSNSQRW KQDLDSKLTL PNGEPVPCLL LANKCDLSPW  61 AVSRDQIDRF SKENGFTGWT ETSVKENKNI NEAMRVLIEK MMRNSTEDIM SLSTQGDYIN 121 LQTKSSSWSC C

The invention provides for a nucleic acid encoding a LRRK2 protein, or fragment thereof.

For example, the human genomic nucleotide sequence corresponding to the sense strand of the human LRRK2 gene is depicted in SEQ ID NO: 9 (144275 bp). Sequence information related to LRRK2 is accessible in public databases by GenBank Accession number NG_011709.1 (nucleotide).

SEQ ID NO: 9:      1 gcgctggctg cgggcggtga gctgagctcg cccccgggga gctgtggccg gcgcccctgc     61 cggttccctg agcagcggac gttcatgctg ggagggcggc gggttggaag caggtgccac    121 catggctagt ggcagctgtc aggggtgcga agaggacgag gaaactctga agaagttgat    181 agtcaggctg aacaatgtcc aggaaggaaa acagatagaa acgctggtcc aaatcctgga    241 ggatctgctg gtgttcacgt actccgagcg cggtaatcac ttgaaaataa actgtgcttt    301 tatttttgca aactttctcc ccctccttac atttgcaaat tttgtcctcc tccccttgac    361 cctgctcaaa cccggactct taaggagccg caaactccca tatcctttcc ttagggcaga    421 aagcagctga gaatttcagg aaggtcttca cctttttgac ttttctcccc gtttcagact    481 aaaaaggaga gggggtgctg tggattgtga ctttgcttct tttccccacc cacttgtttt    541 ccagcctcca agttatttca aggcaaaaat atccatgtgc ctctgttgat cgtcttggac    601 tcctatatga gagtcgcgag tgtgcagcag gtaaaggcat tgttttcact tcaactcatt    661 ctcccttctg tttggaagga gacgttttac tggcaatgtt aatatagccg agagttcttg    721 gttattccca aaatttggct tgaggaacct ctgactgtga ttttaagatg ggaatattgt    781 taaatcatta cgcaatgtaa acgggatgaa gagccccagt atgtgttccc tgagtgtctt    841 taagaagtaa ctttataaaa ccaacagtat ggatggtggt agaaggagga taaaaatggg    901 ttcggtttta gtctcgttat tggcaagatg aattcattag tgtttagact tgactattcc    961 aagtatcttc ccaatacaga gcatgtccta gatgagaaga ttatgaatag tttggaaaag   1021 gggaataatt aatagtgata aaatgcaact ttgtcactag caaactcttg tagagttcag   1081 cactttttaa aattcaaaga tttctagcct ttagttgtag tataccttgt agtatctaaa   1141 gaaagtgatg tcttatgaga ccctcatagt ttgcaactgt tgtcatataa aatgcatgta   1201 gaagtgaaac ttttacaatc tgtaccatag gaaacccaga aatttgctat gtatcttgga   1261 ttttttttta aagggggcct taaaaatggt aattaagaat gatttacagt caaaacaaaa   1321 ttataggcca aggtgataac ttccttcgga gcacttagag atttggggaa ctgaaatcag   1381 ttttgtcatc tgcatgttaa ctcatgcaga gaaagagaat tggactttga actccttgga   1441 ggtgcagtca gaaagccaat gtttcttaat ggttgagagg cttgacagac atgaggcatc   1501 tcaatcttta aagtggtgtg ggtctatctt tatcttgatg tttatctctg tatctagctg   1561 tatctagtct gggtgaacca tctagcttct ttgatatgag gacatttaca tctggaagaa   1621 atattttaat ttgttttcaa ctgtgaaata ttttccatct gactattata gattttcacg   1681 ctgctatcaa accaaaccaa gaaaagatgg aggcataata aagatgctgt tcttttaaga   1741 ctcaaagtcg gaattttgcc tgtggaatat gagtcacttt ttgggcactg gcctattgtg   1801 cttcctgctc tgcacccacg tcatcccttc ttacttgtct ctgctttggt gttcagaagt   1861 gcctgattct ggccaccttc attccctaga ctctgtactt gatagagtca ctcctgcttg   1921 atactgctca ggacagtcag atcctgggta ggcgttttgg tctgcagggt ctagataagg   1981 cagtgctata cttgacaacc caggggagcc tggaacatac ttcctaattc ttaattttag   2041 aaattgccca agcctgagca tacttgtccg gagtagttat gagtgtcact tagtatttct   2101 gcctagagag taccagaggc aaagtatgct ggaaaataag gaagagtttt tttaaaagta   2161 attaattact tttttggata tatcatagtt gtatatattt tggggataca tatgctattt   2221 gatacatgta tacaatgtgt aatgttcaaa tcagggtaac tggaatatcc gtcacctcga   2281 acatttttct ttgtgttggc aacgttgcaa tttctttctt ctagctattt taaaatatgc   2341 aatgaattat taaccataat ttccctgcta tactattaaa tattaactta attgcttgta   2401 tctaattata tttttgtaca cattaaccac cttctcttta tccctcccca tcctttcatt   2461 tccagtctct ggtaaccacc attctactct cttcctccat gagatccacc ttttccgctc   2521 ctacatatga gtgagattat gcaatatttt atttctgtac ctggcttatt taatttaacc   2581 taatgacctc cagtcccacc catgctgttg caaatgacag gatttcattt tttatgactg   2641 aataatattc cattgtgtat gtataccaca ttttctttta tttttagtta agtaattaat   2701 ttagagacag ggtctcactc tgttgcccag actggagagc agtggtgtga tcaaagctca   2761 ctgcaggcct gcaatcctgg gctgaagtag tcctcctgcc tcagcctccc aggtagctag   2821 gactataagc atgtgccacc atgctcagct aatttttttt tcttttttta ctttttgtag   2881 agatggagtc ttgctatgtt gcccaggtta gtttcaaact cctgacctca agtaatcctc   2941 ctgcctcggc ctccatattt tctttgttaa tctgttgata gacatataag gtgattctgt   3001 attttaacta ttgtgaacag ggctgcaata aacatgggag ttcagatata tctttgatat   3061 actgatgttc tttttttgga tatataccca gcgatgagat tactggatca tatgaaaatt   3121 ctctttttag ttttttaaga tacctccata ctgtgtttca tcatggctgt gctactttat   3181 attcccatca gcagtgtacc accattcccc tttttctgca tccttaccag catttgttat   3241 tttttgtctt tttgataata gccattctgc ctgtggtgag ataatatctc attatggttt   3301 tgatttgcct ctccctaatg attagtgaag tttaggattt ttttttcatg tacctgttag   3361 ccatttgtct gtcttctttt gagaaatgtc tatttggatc ttttgtccat ttaaaaataa   3421 gacttttttt tttttttttt tttttgctaa ttgagttttt ttagttcctt atgtattctg   3481 gttgttaatc ttttgttgaa tggatatttt gcaaatattt tctccctttc tttatgttgt   3541 ctcttcactt tgttaattat tttctttgtt gtgcagaagc tttttagctt gatataatcc   3601 catttgccta tttttgttgt aattgcctgt gcttttgagg tcctacccca aaaatcattg   3661 cacagaccaa tgtcctgtag catttcccca gtgttttctt ctagtagttg catattttca   3721 ggtctgagat gtaagtcttt aatccatttt gaaatgattt gtgtatatgg tgaaagctgt   3781 ggatctagtt tccttctttt gcacagccaa tatttgattc tcactgaaat ctcactgccc   3841 tcctggaaat attaccggat gttttactgt catagcgaag tgagagtaag ctgctcactg   3901 aggatcaaag agcttgtgac agacctaaga ctcaagtctt ctcacacctt caaaatctct   3961 ttccatcata caatctacta gctgctgaat tcgcaagctt tttgtgcaag ctagtaaaaa   4021 gcaaaatggt ttgatacaaa tactgtggcc atgctaggta caatgacatc aatttaaatt   4081 atcattggtc ttaacaaggg gatgtagaaa ggggtctcct actgacattt taatactcac   4141 ttaaaagtag tatttttcct tcagatttct ttatattatt agtataatta ctgtaagtat   4201 cctttactgc tttatatgtt gaattagctg gaagtgccaa aagaaaaact cttaatgata   4261 aatttaaggt attaaggtaa atttctcctt catttaattt aatagtaatt cttaattact   4321 atttaaaata aagattaagg tttgtttcta gatgccattt aacatgatat tccagactgc   4381 cagttttatt ttcaaagttt gtttcatatt ttattaatgt ttcttcataa atgacagtct   4441 ttagaaaatt gacggttaag ctaggtgctt tatatttttc ttttcctgcc tatcttttca   4501 ctgtgctcct aaattttaca tctctttatt ctcaagggtt caacctttga agaaggggag   4561 caaaataaat gaaagtggct aaaatttttt ctttaacccc tagactcttt cctgttgtgc   4621 attaattaca tgcttgagtt tttagaataa ttataataaa gtaaaactac caatttaatt   4681 gtattgtaac tgtgcaagat gggaaccttc tctcttagag agataagctt ttaattgaat   4741 agattaatgg atcaattgtt acctctgctt tgctgccaga gattctattt aatcacagaa   4801 gttccatgta gtgctggaga gctcagttgc ctgaatcttt ttgcaaagcg tttactgata   4861 ctgttgcttc accaaccaaa acaaacaggt tttttccttg agtcagcttt gtaggtacag   4921 agatgagttt ggcatcctat gtgacttttt tttttttttt tttttttttt tttaccacca   4981 gaagctgttc agaatgttat tttcttaaat agttcggaaa aaagtcttga tgtattctat   5041 gaaagcacaa aaatagtcag tttctatgac agctggattg tcaacgtctg ttcagcttac   5101 gtggaggagg atgtcctact tgagtagtat aggtagaaat agctatcaga aattgccgcc   5161 tttgaaagca atttgaaatt atgtaaaagg aagtaatgac aaaataaagc aatttatgtt   5221 taatctggaa aagatccaaa agtaatattg taaagagatc ttgagtaatc atttttatct   5281 tcctaaaata gccgttgttt actcccgtaa gcgagtaaga aacttgtgcc attattcctt   5341 attgggtgca tatagatttc tcaccttgtc attcaactcc ttgcaatatt caactttact   5401 tatgcatcca gccttatccc aaaatagcct cttccctgta gcagcttcct tatcatgtag   5461 cagcctactc tcctcaccgc tcatccgttc ttatacaatc tggcttaagc tctacctctt   5521 catattatat tcttctctga gtaatttcaa ctcacactga gtcttacttt cagtatttct   5581 attatattgg tatttattac acatcacact tagatacttt tccattagtc tctagagggt   5641 acatatatgc actttctctt tttttttttt tttttgacca tgtcaagtat agttctatag   5701 tataatagaa tgaattggag atcctttaca tttagagagg gaggagtcta cagtaggaag   5761 aataggttaa ttttcatctc cggtttgaaa tcaggacttt caattttttt ttcagaggta   5821 aagagcaact tagtcaagtt ggcatcttgt aaacagactg agtgaagata tacttaaaat   5881 gcatctataa tttcatattt tatttcgaaa tgtgaaagag cctactaggg gtgtctgtga   5941 tctctagacc ttatcaattc attctagaga aatctggagg gagccattga ggagttctac   6001 ctcctgtcta ttttatagag ctctttctct ttttctccta tcagatgtag attcaattgc   6061 taaaaatgcc acgtttcttg cctctattat tctagcttca ttacttgggg agcagccatt   6121 ctgataactt acattttgct actaaaatct ccaacttcac ctaatccttc attataagcc   6181 acttcatttt ttcctataat taaaatttta aatatgtgga ggaaattctg tcaggtagat   6241 atgacttaaa acctactaag ggccaggtgc agaggctcac gcctgtaatc ccagcacttt   6301 gagaggccaa gtcaggagga ttgcttgagc ccaggagctt gagaccagcc tgggcaacag   6361 agcgagaccc ctgtcttcac aaaaaaaaaa aaaaaaaaat tagctgggtg tggtggcaca   6421 tacctgtagt cccagctact tgggaggctg aagtgggtgg atcacctaag gacaggagtt   6481 ctaggctgca gtgatctatg attgcaccat tgcactccag cctgggtgac agagtgagac   6541 cctatctcaa aaacaaaaac aaaaccaaaa aaaaaaaaaa aacaagaaaa aaaaagtact   6601 aaggatctgg tatagccatt cttgcactta ataatcttgg tacaacctct aaaactattt   6661 tttatagttt attttatttt gccttattta gacaattggc atgtctatgt tcttcataat   6721 ttagaaatta tatgtattta tatatataaa ttatatatat tatatattat gtataaatta   6781 aattatatat atatataata tatatatatt tgaaatggat tcttgctctg tcatccaggc   6841 tagagtgcag tggcacaatc ttggctcact gcaccctctg cctcctgggt tcaagcgatt   6901 ctccttcctc agcctcccga gtagctgaga ttacaggcgc ccatcaccat gcctggctaa   6961 tttttgtatt tttagtagag actgggtttc accatcttgg ccacattggt ctcaaactgc   7021 cggctgaaat gttgtatttt ttgtatgtct ttctggtatg atttttggag aaaggtgtat   7081 cctaagaata cggcttgctt ttgtttctgg gtaagcattt tagggtatca ttttgttgta   7141 taaccattgt ttacaagtga gataagcatc tattccacta agattgaaga gattcatgtt   7201 tgactgagta tgctctatta acattcttta aaacatgtga atatatgtct ttcttgtttt   7261 caggtgggtt ggtcacttct gtgcaaatta atagaagtct gtccaggtac aatgcaaagc   7321 ttaatgggac cccaggatgt tggaaatgat tgggaagtcc ttggtgttca ccagtaagta   7381 tgatagatat gtaaaacaaa tggccttgag tatttatttg tacacatgac aaccttccct   7441 tgatacactg tgtttgcaat ccaaggctac tcctgtggaa ttctttaaaa tacagatatt   7501 tttccttgag tcaatgattt acatttatag agagctttaa actcagaagt ttgatttaga   7561 aagcaaacat ttaaggtaac atgtcagaag ttattatttt aataatataa tcatataatt   7621 ataaaactgg ttaagttgta gatttttgat gagtactttt gaattcaaac catgaagaga   7681 ttttggcttt taataataga atcgatacaa accactagtt cttaaaaaaa tgggaactga   7741 gaaaagttag ttctgtaagt agtaatttga aagttgatgt tctactgtct ttaaatagta   7801 catttatata tatattccta tatatacagt aagtttaaac tatggctttc agaaagagtt   7861 aagaaagagg aaattaactt tcagcacatc tgtagccaaa tcttgatagt aattttacca   7921 gctatgtttt tgcagtttgc agcataatgg cttcttagat gagactactt ccttagccat   7981 cattaattaa gaaaatattt tctcaagaag aatgtgtttc caggaaaata cattttggat   8041 agctttgttt cttgacagtt aaaaaatatc ttctaagcta ctgaggaggc tgaggaagga   8101 gaatcacttg aacctgggag gcggaggttg cagtgagcgg aggttgcagt gagccgagat   8161 tgcaccactg cactccagcc tgggtgacag agcgacactc tgtctcaaaa acaaacaaac   8221 aaacaaacct tttgtcatta actttaaatc ttttttatac ctaatatgac ttttctttat   8281 cacagaaaag gaaattgtga atattttttg gcttccaatg gtatatggtt tatgaaaatt   8341 taatttatga aaaattttca ggtgtttgta ttgctgatca gtgtcaagta gtgctataaa   8401 tttagacaaa ttagagctat gtgtttgtcc ataagtgaac atgtctgtgc ttatacattt   8461 tcccctcttt gacaaatgtg ttgctcttct tgttttcagt acataaaggg tgtgttttgg   8521 aaagagcata tttacaatta attggagttc tcgtcttcaa tctaatctct gtaattctat   8581 gtatcagttc taaagtatac agcatttgat gaggaattac tcaaaatata ccagtaatta   8641 ggaattgtaa ctttaaatgt cccttggttt gggtgataat ttccaggaag tccaaagatg   8701 agccagtcta taacctcagg gagtgtttgg gaaactcatc tagtcatatt cctgtacaaa   8761 ccaactgttc aaattaaatt acataaaagt ttatgtagga aatttcattc actcactcac   8821 ttactcattc actcactttg ttcatccagt cattcatctt ctattcattg aatgtttttg   8881 aagcctgtcc tctgggtcag aaaccatgca gttgtgaaga agatagacac actgctgtct   8941 ccagtggagt gtattagatc actcccagca aaaattgatt gtaaaacaga tttctctttt   9001 ttcaaggcct tttccctcca aagacttacc agtactgaag aaaaatttct tccgtggtaa   9061 taaagtcagg aattgtggga atggtatagg gagaggtagg ggcagggtga ttaggaggaa   9121 ggctggcaga gaatcgaaga ctggcttcat tcaggtcctc caattgccaa atggagatta   9181 tgcaacgttt cttgaataca tacaaaactc tagatgtggc cagctcagtc ttcttccaat   9241 aatgtaaagc caaacaatgc tttgcaggaa tagactagag attatatttt gggattaata   9301 acatagggat taaaatctta tcttgaacta actaaacatt attgatatgc taaattcact   9361 tttttttttt tttttttttt ttttttttag acagagtctt tctctgttgc caggctggcg   9421 tgcagtggcg tgatctcggc tcactgtaac ctctgtgtcc cgggttcaag tgattctcct   9481 gcctcagcct ctcgagtagc tgggactgca ggagtacgcc accacgccca gctaattttt   9541 gtatttttag tagagacagg gtttcatcat gttggccagg atggtcttga tctattgacc   9601 tcatgatccc cccgcctcgg cgtcccaaag tgctgagatt acaggcgtga gcaacggcca   9661 ccggcccact actttttaat atatcattaa tttctctttt aaaaacagta gcaatcaata   9721 atttaaatat tcaaatgaat tcttaattta tatacacaaa ctaacatctt tattatatct   9781 ctatatttta atatatcaac atgtctaaga taatttataa atttacatca tatataaaaa   9841 tgggtttgct ctcgatgtat ataaggcttc atgatatttt gaatatggag ttgggtgaaa   9901 atagtgaatc tgaatatttg aatttgaata tttattggaa aataagtagt gcttttaact   9961 ttttaaatga gacacataat agtcccctgt tgattttttt ttattttttt aactttatta  10021 aagtatagtt gacaattaaa aattgtttat atttaaggta tacaattgat gatttgacat  10081 gtgtacattg tgaattattc accacaatca agctaattaa cattccctgt tagtttttat  10141 aagcctggtt caggtttgta gaaagaaaca aacacacatg gccaggcacg gtggctcaca  10201 cctataatcc cagactttgg gaggccaagg caggaggatc acttgaaccc agaagtttag  10261 accagcctag gcaaaatagc aaaccctgtc tctccaaaaa agaatgaaaa aattagcctg  10321 gtgtggtggc atgtacctgt atccctagca actcaggagg ctgaggcagg aggattgctc  10381 acttgagccc aggagtttga ggtttcagtg agctatgatt gcaccattgc attccagcct  10441 gggtgacaca gcaagaccct gtctctaaaa gcaggcaaca aaaacacatg agcttcacta  10501 cagggaatta aatacaatga gagtaataaa aaataggtga gcaaaaaaaa tgcaaataag  10561 caaacttttg agtatgatat ttcattctta tcttgatttc tgtttttaac tccagattga  10621 ttcttaaaat gctaacagtt cataatgcca gtgtaaactt gtcagtgatt ggactgaaga  10681 ccttagatct cctcctaact tcaggtaata tgtgtatatg ttttttgtgt tgattcaaat  10741 taaaaaaaaa gttgatacca ttaagtaaat gtgtgtgtgt gtgttttttt tttttttttt  10801 ttgaagatca ggattagggt agcttgattt aaatgtccta aaattgcatc tgtttttaga  10861 cctagtgatg ggacagccat aatataatct aaatatcagt tatttccaaa attctttctg  10921 tttccatctc ttctccttat ctcttttctc tatactttgc ctctcaaaaa tctcattcaa  10981 tacattggtt ttaaacatta ccttatatat tatccccaaa tctctgttgg tagtccgatg  11041 tttgctccca aaatctggac ctacatctca tattccccca ggttagtggt cattcctgcc  11101 cttgccatta ttacttcttt ctccctatat atctttaata aattttctat aatatatgtt  11161 ctggagtatg ccataatcgt tacattttga aaacacatag tattacttct tgagtatttg  11221 ctagatgcca ggcttcacaa gtaaaatgct tcacgtgctt ttaaacacct gaatctgaaa  11281 acaccccttg agatagggat tttatctcag ttttctaagt gacaaacact gaagtgcaga  11341 gaagttgctt tggccactaa gaggtagaaa cagggtctga ttctccatgt caggttcctt  11401 ccctgagaaa actttggcct ggtagataat ggacctgaaa acaaaaaatc ttgaaatgat  11461 gcaacagttg tgggcattgc tgtgctggac actggctatt atataaggtt ccgagaagaa  11521 aggccgctca cagggagcta atcttgaagg gctgggagga gtttccttcc atgtagggga  11581 gggcttttta ggttgagaga agtatgggtg cagaggcctg ggaggatagc atgagagagg  11641 ctggacgtgt gattgggaag gtttgaattg tcctcatcag tcctgctaag agatgtaaag  11701 accatgctgg agaaagagga agatgagagt atgagggaaa aacaagaagg tactcaacat  11761 ttcactacag ctttttatga ccatgttgta tggcatgcac taagagtctt taaccatgat  11821 ttaatttaac ctcacccttg ggaggtattt tttttttgac ggagattcat tctccatttg  11881 gcgatggaca ggaagatgag ggtttattaa tatgaaaaat ctaccaacac tggaatatat  11941 tgaagttagc ctcatacagt actactactc ctattccagt attattattt ttattgacag  12001 aatagatgct gtttgtgtta agttttggat tatgatagga aatgtttggt atagtaaaag  12061 gcaagagtgt gacatgcagt tagtccaagt acgaagagat accaaaaaaa aaatgtttag  12121 tgaggagcag agtttagcat atttggagtg aagacaatgt gcggaaggaa aggagctgat  12181 gagatacatg tactatagtc ggttgtgtga aaggtcttgt ttttcatact aaggatcatg  12241 agaagatctt agtagattcc agcaagggat ttgcaagacc acatttgtgt tttagaaata  12301 taatgcaggc aataaaccag ttggatagaa attggggact gtagagcaat taagcaactg  12361 tttttgcatt ctagatgaga atgcaaaaca caataggaat gaaggtgcct tgttcaaaag  12421 gagttttgtt caaaaggaat cttcaagatg tgtaggagat attcttaaag gacttggtaa  12481 tgaattgatt tgttgcttgg atagagaatg agaagagaaa ggagggtgga agaaggaaga  12541 tgacttagga gtttctcttg ggtagctagt ggattatggt atcattgatg aagacaggga  12601 acaggagtag gccaggtttg gggtaactgt ggaatattca gatgttgtct aagaggcata  12661 agaatgtatt tcagatgttt ggggcaagtt gtctaggcta gaagtactga ttgagactca  12721 tgaaattata gtaaagtgaa ctgggagttt atctcattta taaagatcta gagcttgata  12781 agtctaacat ctagggcagt taagtagttt atcaacaaac aaacaaacaa acaaacaaga  12841 aaaccatggg tctacaaacc attcacagtc ttcatgtaaa aattaattca tgtaaaaatt  12901 aacacattaa atgttaaagc agctctttac tcagagcata ttattctctt taaaataggt  12961 aaaatcacct tgctgatatt ggatgaagaa agtgatattt tcatgttaat ttttgatgcc  13021 atgcactcat ttccagccaa tgatgaagtc cagaaacttg gatgcaaagc tttacatgtg  13081 ctgtttgaga gaggtatttt aaaatgtcaa attccttaaa gtatatataa gaaaaaaagg  13141 cttatactgg gaaaagtaga acacagttat aataagaaga aggtttctaa aatcctacta  13201 tttattaaga agtgggagtt gtctgtcaag ggtgaggaat ggggttaatt cagaagtatt  13261 gcttgttttg gtggggtgaa tttcattcgt gggttataaa tcatgcccct ggagtagact  13321 ttcttcaatt gcttaacaag gcataaggtt tactttgaaa actggatgtg tgggtgctat  13381 gaaagaaaaa ataaaactgt gaagccaagc ataggttaca ctgggattat gatgttgagt  13441 catcaccaga aatcatagaa attgcataaa gagcctgaag gtttacaaag tgtccttcag  13501 gaaaaagact aatatgcatt tcatagcctg gccctgagat tgataactga gattattatg  13561 taattttaga gttggttgga gtccttgttt agtctttcca ttgaccttag gaggaagtgg  13621 gtcacagcag tgaagtgagc atcctgcctg aggacacaga gcttgtgaca gtacagttca  13681 attagcaatt attttaagag ccccttttgt atcattatga gagccaactg tgctaggggt  13741 ttagataaga atgatttatg tgggccctgt gtcagttatc agtttaccag tctaatttct  13801 tgcagttccc agaatgggat agatcacctg ataactgttg aattccctgt ctcctcccag  13861 aaggatttta aacagcttat agataattat aatacacaag agtaaacaaa atggatgaga  13921 aaataggtga agggacaata atataaagct agattaagtt tactgtgttt ctaaggtcct  13981 gcatatttac aaaggggtgg gccagaaatt tgtctgtttg cttcctatct gacaaagaaa  14041 agaggtaaat atcagtggtt acaaagttcc ttaagataaa agtaaaccta ttattcagga  14101 gaagcaattg gtctcatggg agatctgaga aacatcttcc catgggtttt cctggatgag  14161 acaataaagg acatacattt tgcaaggaat acaaagtgta ttgcagcgag agtgactctg  14221 tcaaaagtca gaatagcatg ggcctggtac ccagctcttt gataatcata caccgtgaag  14281 tagaagatag tttacagcga gtacggaatt ccttcaggct gtcatgtata aatgttctat  14341 cttgcaacta agctttcgat gacaattagg ataaagtttg aggttctatt gtcttgcagg  14401 gtctgtaatc ttctgtgtgg aaggttaggg gcacattctt cttcctggaa ggagggctag  14461 catcacttta tcaccatcgt tgtttagtcc atctaagaca ctggaggtag accatagaat  14521 gttacaaaga agaatgttgc tcaatagaaa aaccatcagt gctgagaggg ttatgactat  14581 aaatgtagag tagaaaaatt tctgattttt ccaggagtat caggttctcc aggactcagg  14641 ggtgactata aagttaattt tcaaaatttg aaagtgtact gtggaaacta gaccataaag  14701 tgagaaagtt ccatgatatt cttcacttgt taggaaaact taactgattt cacattatat  14761 tatagggaca ctctggcata aaattaaaaa aaatgaatgt tgatcactta gagtgctgtg  14821 ttttctaaca tatttctggc gccattctca agctagataa actataattt tatacatgtt  14881 tttcaggttg ttgcccaata acaatgactc caaatggaac ttactggctt gatcaaatga  14941 ctttaattgt gaaaattaat gatttatatt tttgctgtct gatggaaaac cactaagaca  15001 gagtatttca aagtctgatt acttgccatt tgctcaagtt gacaactctt gaactgaaac  15061 atttagccga gctgcccttc agcagcctac cattaatgcc tcccttttaa atattgcaat  15121 atgtccagtt ccagttggcc atctttatta gtcactgtca gttttctcta gaatttccca  15181 aatgaaattg taaataattt tgtttttctg agaactgctt gctgactagc acttttacat  15241 ttcaaaacat ggagtaccta acataggccg aaacaaaatt atttgaatct ccgtagcttg  15301 ttttctcatt ataacattct taggaagggc tgcttcacag aaatatattt tttatttaag  15361 gagattacac ttgatgtatc tcacacaact ataatgaata ttgtaatttt tgaataatta  15421 aactttcata tcatctttaa gcttattcag tattttgtct ttcattttta agtctcagag  15481 gagcaactga ctgaatttgt tgagaacaaa gattatatga tattgttaag tgcgttaaca  15541 aattttaaag atgaagagga aattgtgctt catgtgctgc attgtttaca ttccctagcg  15601 attccttgta agtagcattt aaatgttatt tattttttgt atctgaaaaa ttacaatata  15661 tctcattctg agtatatttt aacaatattt ttattattta gaaacttgtg gatgctcaac  15721 ccattcattc atttattcat ttaattaatt tacattcact gacattatac tgaagttggc  15781 tgtgggcttg gtgctggaga aacaatcgtg gaaaatacag atgtgttcct taccttttca  15841 gagcttgtag tacaatgggg gacacagata agtacagagg tgattacagt ggcagaagtg  15901 atggcagatg gcagaagtac ctagagttag gagatcaaat aggaagtgag gcagtgtctc  15961 ttagcaaaga tttaataagt ggagcttcct gtgcatgaag gtgtgacctg aagtgagaat  16021 gcaggcaaag tggcccaggc agtgggcatg gttaatgtaa agatgctgga gcaagagaga  16081 gcagactgcc ttcaagaaga caaaagtagc tcagtaaagg tgtggggtta tgagtgtgcg  16141 tgcatgcatg cgtgtgtgcc gctgtgcatg cacatcccca aatatcctat ccgtttgtgt  16201 ttcattgaca gaggcaaggg agagcttgat aagaggcagt aaatgaggcc agagacatgg  16261 agtggagagc atgaagggcc taaaaagcca catgaaggag tttgaatttt attgtgactc  16321 ttgattagca ttttaatgag gctttgaaat ttagccacat ttttcaccaa aaatattaat  16381 cagaagaaat taatttgatg tgtatgctac caatgattgc tattaggcta aaataatggt  16441 tcatattctg ttttgttttg tattaatggt tcatattctg ttttgttttg taagtgacca  16501 ttaacacttt gtattttatg tattacttgt gtgggtttct acaggatata catatgcatt  16561 tatctagtga tattttcatc ctcacacatg tgaagttttg aggattagag ttaaacaatg  16621 tacctggtat gtaataagtg ttctaaaatc actgacagga ttattagaca atatgtattt  16681 tatatgtgtg ttgtatacta tatgtaattg catttatggt ttcagatatg gaaatcactg  16741 tgtcaatctg aaggtgtgag ccttcggtgt aggcagagta aaacccaatg cccttgtgaa  16801 agaatgcttt tttttggtga tgtttataaa atcacaatgt tttcttatcc acaggaaatt  16861 aaacactgga aagtgggtgg ggctgaacaa taatagagaa aggccatggt tttacatttc  16921 tctgagacat cactgccaac aaactgaata tgtttttcat tatacttttt ccttggctat  16981 atttattcat ttatttattt attttgggct ggaggttttt ggaatccatt gttttccacc  17041 cacattggac ataactccag taaaaatgtg ttgattcata atgcaaaagt caagaaagta  17101 gcagctaaaa attaagaaat caaaagtttt taaaacactg attctaactg aaaaacattt  17161 gcttttcagt ctttaagtct attgttctga gtcaaagcag ttcatttcct tacgttgtta  17221 attttttttc tatgtttaag cattgtaata tactttttgt gaaaacagtt gattagtttt  17281 ggttgtgcca aaacaaatac taaaatgttt tgcaaacagc ctttttttaa acaaaaaaag  17341 aacagttaac atttgatgca gagatataca tgttttctcc atgtaggttc acacctcact  17401 tcctttattg attaattgct ttttctggta gagtctttct ttcctttctg ttttacctgt  17461 gtttgtccct aagacttata ttttaatatt atgcctcctc tctttcgttc tcccatcttt  17521 tcttccacct attttggagc cttcaggaag cttgattttg ctgccttgta cattggttgc  17581 ccttctggaa tggaggaaac aggtcatagc tgattttaac tgttccatct ggtgacatat  17641 tcttgatttt ctttcttttg gttggggaaa aaaaaacaat gcaaaagtca ttctccaatg  17701 gggttgagcc tcgttaagaa atagaccctc cacaatggtt gaactagttt acagtcccac  17761 caacagtgta aaagtgttcc tatttctcca catcaaaaaa aaaaaaaggt aagcaatata  17821 acatgagcca tatctaatag gacttcagaa attatctatc ctatagttcc aggatgacga  17881 tgatgatggt tgtgataatg atgaagattg tgatgatagt tatatgggag ataaaacttt  17941 aagcacttta catattaaat tctataatat tcaccacatc tattaaaata tgttacatta  18001 ttgtccctat tttaccaaca agaaaactga ccaacaagat taaacaacgt gactaagttc  18061 acacaacctg taacagcaga atctatgtca atcacaacac aattagcagc tatttctgtg  18121 gcaattttca ataaagatgt gtctggaaaa aaaaaaaaga aatagaccct ccaatttatt  18181 tatctgaaaa cttatgacca atacattaca tttccagact ttcattttca gtacttttcc  18241 tttcattttc agtactaaaa gtactctgaa tttttccttt ttttgatctt aaggctttaa  18301 gccaagaaac aggaataaag taaattttcc ttaatgccaa agattagtcc tacaccccat  18361 tatgttatta atgaacagca tagtattttt tacagctact taaagaacat gatgtttaaa  18421 tttggaaatg cagtcattat gctgccatct atttacagtc tatataagac gtctttgtat  18481 gcatatttga aaggagaaca tggttacctt attgataatt atgatctctt taaattcagg  18541 caataatgtg gaagtcctca tgagtggcaa tgtcaggtgt tataatattg tggtggaagc  18601 tatgaaagca ttccctatga gtgaaagaat tcaagaagtg agttgctgtt tgctccatag  18661 gcttacatta ggtgagtttc ttagttaata tgtcatcaca cactgtatga tatacatata  18721 catataaaac atatatatgt tgcataataa tggataagta gcatattgac atactttgaa  18781 tgaaaatatt gtaaaatccc agaaaaaata aattaaaaca aaaagaaaat actgtaaatt  18841 accaaactgt tctgctgtgc ttagatggac ttttaaaagg agtgtcaaaa atagatgtgt  18901 agaatgtaaa agaagtatta tcttaatctt atttttatag atgtagttct atagatgagt  18961 ttttttattg ttgaggctat atttaaaata taattatgta agaattgata catacaaaaa  19021 tatgcataac atatacaata taaagcataa tgcaaataac tcactatcca acttaaatgt  19081 tgtatattcc cagtggggga agctaccctg ggcttcttcc tgcccttgct ttctctatag  19141 aggttaacac tatccagaat tttgtgttta caaatctttt gtttataaat atgatttact  19201 acattttcat gtttctctaa gcaaaattgt taatgtttgc ctgcttttgc ttcatcaaaa  19261 tgtaatcata ctgtatgttg tcttctgcaa ttttcaaata tcagtgctat gattataaga  19321 atcagaaata tttttgcatg tggttgcatg tagtattcta ttatttggaa ataccacaat  19381 ttatttttcc atttttctat ccatggacat ttggattctt tcttatttta tgctactact  19441 atctgtgtta aaacagataa ctgaaaaaga acagttaaca tttggtgcag agatatacat  19501 attttctcca tatataaata agggttaata ttttaaaaaa tatttatttg ccattggtat  19561 gtccttcttt gtgaaatatg caatctgtca tttatcaata ttgttcacag gatagtctgt  19621 ctttttaaaa ccgattcata gattctggat aataatgttt tggtggtagg ttatacgtat  19681 tgaaaatacc ttcccttgaa catcacactc tggggactgt tgtggggtgg ggggaggggg  19741 gagggatagc attgggagat atacctaatg ctagatgacg agttagtggg tgcagcgcac  19801 caacatggca catgtataca tatgtaacta acctgaacat tgtacacatg taccctaaaa  19861 cttaaagtat aataataatt ttaaaaaaaa ggagatgaag aggtagctgc aggttgactg  19921 agcaagggtc attgtctatt tgaagtttca aaggtatctc tgaaaataaa cacagttttt  19981 tgcaagagtg aaaaaaaaaa aaaaaagaaa ataccttccc ttgacttgta gcatgtcttt  20041 tcaccgtctt tatggtggct tttgtgatat agttaaattt aataatcagt tcctttgtga  20101 tttactcttt tttatgtatc ttgtttaaga aatcttaatc tgtcctttcc ctaagataat  20161 aaatatattg tatattttat tccaaatctt actcgtttgt taaactgttg cagtttgttt  20221 ttgtaaagaa cccaattccc cctctttttt tcagtgtgga gagctagtta ccatcacagc  20281 acaatttatt agaggttcac ctgtttccca gtcatgtggt atataaatat gtaaacatat  20341 atgcttatgt ttctgtcctt ggcgttctgt tgcattaatc tgatttgtct gaatcagatt  20401 taaatctgat gttaaatata acactattta aattaatagt tctgtaagtc ctgatatctg  20461 aaaagctaat aggtcaagtc accatacatt cttttttagg cacagcttgc cttttttctt  20521 gggcctttac tattccatgc aaattacagg attaacttgt ccagttcctt ggaaaacact  20581 gttgagattt tgactggaat tgcacaaaat atgtggatca ttttaagaag aactgacatc  20641 tttacattat aatttcttct atacatgtct tgcacatctt agtttttgct acgtatctta  20701 ttttttatta ttattgtaaa tggtatttct ttttaaatca tatttttgaa ctgtgttttc  20761 ataaagaaat gcaaatgatt ttttgtatat tgaactaacc tacctttcta aactcttcgt  20821 taattctgac aatttgtttc taaatgcact taagttctct acctagcaat tatataattt  20881 gtaaataatg acagtctgag ttcttccttt ctgcttctta tattctttat ttcatttcct  20941 tgtcttctta catgagtcag aattattaat atagtgttaa atagagccat tgatactaga  21001 catccttgtc ttgctctttt tcctgatttt aaacagaata tttttaatat tctcccatcc  21061 aaaataatgc atcttacaag ggtttaaact tttaaaaaaa attttgttaa gaaattttat  21121 tttatttctt ctttgctttt gttttttcaa tgtgggcttt taaatgtttg cttttatttt  21181 tacaatgtgg gcttaaaaat attaaaatat ttaattttat caaatatact taaaatgtag  21241 taagtctttt ttttcctttt cattatgtta atatgaagaa atacatctac aggttttcta  21301 atactaagct actgtgcact tcctgataaa tttaacttgg tcatttatta gatttttaaa  21361 aacacttcta aaaaatcttc tctgtttatt ttcacatata ccaggtgagt tttggcagcc  21421 tcttgttttt tcttttttac cctttctttt ttgttaaaaa catcctttta tttatttaat  21481 gattaataca tggctatttt gtgttttgta tctgataact atatataaag ttctggggag  21541 tctaaaccaa ctgcttctag gctgactgcc tgtcagttgc ggagccttgt tttcttgtat  21601 ggttgtgagt ttctctttta ttgagcatcc tgaggactta aattaaagat gctctttcag  21661 aggtgtttgc caggagtcag agcacaggac tgacctggga gcagtttagg atatgatcca  21721 ggcttaatat gggagactct ggttgagacc ttaccttgca gagagtctga aactggtttg  21781 ttgaatgcag cgccaggatt catgctttcc cacaagacta ctctggcgtt caactcacag  21841 ctcttgtttc agcttctttt tgaactccct ctgcccctca acacacacct tggaaatttc  21901 cttgatattt ttgtgaggac aacaatgcat ttaaaagtga gagtggttgc tgaataataa  21961 ggaatgatcg ttaactgttg gacattgatt ctgatgacat tttctcaaaa ggataaccag  22021 gaatattttg tacaaaatta ggattattat aataggcatt tagtttttca ttgttacaaa  22081 ttttgaggaa aatcattaat catttgaaaa aactaattga catgtctcca ttgtagcaac  22141 ttgtattttc acttctagta tcatgattat atcccctgtg taatttggaa attgatttta  22201 gcattaggaa atttcctagt ttcagttaaa atgaattttt tgtaagctga attctatttt  22261 acatgcacaa ctttagtttg ttatttcttt ccttgacaag catttattga atgttgttat  22321 atgactaaaa ctgtaagatg ataatgtttt aatattttca catttctttc atagcttcca  22381 aagtgtatat atatactcac atacttcata tatatgaatc tacttatata ctgtatataa  22441 aaatatgtat ataaatatat acacattgta tataaatgtg tatatatatt tacacatgta  22501 tataaatata catacaaatg tatataaata tacatattta tacacagatg tatataaata  22561 tacatattta tacacagatg tatataaata tacatattta tacacagatg tatataaata  22621 tacatattta tacacagatg tatataaata tacaaatata aatatataca tttatatata  22681 aatatacata tttatacaca gatgtatata aatatacaaa tataaatata tacatttata  22741 tataaatata catatttata cacagatgta tataaatata caaatataaa tatatacatt  22801 tatatataaa tatatatatt ttttgatacg tacaactatc ttgggagatg ggatattgtt  22861 attatttcca catttacaga tgaggtcctg tggttcatca atctttgtgt tttattcaag  22921 attatgtaag cagtaaggga tgaaatcagg ctgagaaccc aaatttcttc atccctaaac  22981 aaagtatttc ttctaaacat ggtatccatt tactagttta tctctatcag gtgacccttt  23041 attcattatt tttcatgaga aggttgtagt tgtacaaagt ggctgatatc tgataatgtt  23101 ttaatctaat tcaaagtcga tttcttaaat ccaggtgtca gagtagcaca ctactgtaca  23161 attctctgtt tcatgtttta caatttacca cagtcaagtt acaacttgca catgttacat  23221 taaaatgtga attcacctta atttcttgaa atgagccaga agaagaagtg gttttgtttt  23281 gtgatcaggg aaatgctact tgactgccaa attgtctaga acagcacatt aaagttgctt  23341 gattttatac tgttaaaatt aaataaaaca tggcaactgt catgtcatat gtaacttttg  23401 tattattctg taactttttt tgaaaataaa aagtgatcaa attgatcttc aggtaaagaa  23461 tcttttttct cttgattatc tcttagtgga tgatgatttg ttccatttaa tgggtagaga  23521 atttattgtt gctgttattg tttcaaaagt agctgaatga aactcttaac tttttcttca  23581 tagttaaaat taaaacctca agtaaataaa tatttaacgt tttgccaaac tatgtaatct  23641 aattatgggc ttaatgcatt taaaggcttt gtataatttg ctacgtattt tcacacaagt  23701 tacctgaaca taagtccagt cttcctgctg ttttctgagt cacacagtga ttcagtgacc  23761 gaacagctgt tggcagtggt gtgggtataa tagggaaaag agactgatgg ggacaaccca  23821 agtttagaca agctggtaaa agtagaagaa aatcttcttg aaaacactag tgatcactaa  23881 gggctgtgga gaatttttgc ttggtgggtg aatgtggaag aggcaacagc atgggaaggt  23941 gttggtaaag gagctccata cttgcttaaa ctgccttttg attgtgaggc cgttgatgaa  24001 tatttagttt gggctttagg ttttttatga tacaggatat tttccatttc tggctttgta  24061 tctcagagat cacttagtta cacttataga tgaataggag tttcaattcc ttgttttaga  24121 aagaagcttg gtaactgtta gtgagttaca aataagccaa atagaagaag gtacatattt  24181 ctgcagtatc aggtaaagtt tttcctcata aggatttaga ctcttggata tcatattaat  24241 tctcagaaga gtgggtataa aaaggtatgg gacttcttcc tggggtgggt tggaggtggt  24301 gaaatacctt tttttttttt tttctgagat catcatagac aagatcaaat aatggtaaac  24361 atgccaatga attttctaag cactattcct ttaagtgaaa gaagagtgtt tcagtaaaat  24421 gatttaatat tgggtcttcc aaaagatgga tttaagagtt tcaactttaa aagacagaaa  24481 aattaagtta ttttacacaa tgaatattgt cgtgccgtgt gtcacagaca tgacatgaga  24541 gggaatcaga gaacatacag ttaatacaac gcaaactagt atcattactt ttgctcaatc  24601 acttccattg tctaagtaag ataattaaag gacagcataa aataaaattt caaaacttta  24661 ctcaatcata ttaagctatt ttaattaaag taaatgtttt aatgccattg aatattcatc  24721 accattcaaa attattgatg taaatagtgt tatatgttaa aggtaattta acttccatgg  24781 atgagaattc agctaatgtt tcacttaact tttaggtaat tttttcaata tcctggtatt  24841 aaacgaagtc catgagtttg tggtgaaagc tgtgcagcag tacccagaga atgcagcatt  24901 gcagatctca gcgctcagct gtttggccct cctcagtaag taacttcact aaaaagggga  24961 ttcttacaga ggcatttgac atcaaatatg aacattgtaa caagagaatc atatgtacag  25021 atggaagcat tcaatgcctt ttctgtcctg tgtagctcat tttccagtag aggatacttt  25081 caaggaaact aacagttgtg acaaatatac acatctcaat gtagagtttt gctttacatc  25141 attcttgatt tagctttgtc attaagcagc taatctgttt taaaaaaatt tttatttgtg  25201 cctgggcatg gtggctcacg cctgtaatct cagaactttg ggaggccgag gcgggtggat  25261 cacaaggtca ggagttcgag accagcctgg ccaacatggt gaaaccccat ctctactaaa  25321 aatataaaaa ttagtctggc atggtggcgg gcacctataa tcccagctgc tcgggaggcc  25381 gaggcaggag tatcgcttgg aactggaggg taggagttga agtgagctga gattgtgcca  25441 ctgcactcca gcctgggcaa caagaatgaa actccatctc aaaaaaaata atttatttgt  25501 gttttaagtt ctggtgtaca cgtgcaggat gtgcaggttt ttcacatagg tagacgtgtg  25561 ccatggtggt ttgctatacc tattaaccca tcacctaggt attaagccca gcatgcatta  25621 gctatttttc ctaatgctct cccttctcca gtcccatccc ctaacaggcc ccagtgtgta  25681 ttgtccctct ccctgtgttc atgtgttctc attgttcagc tcccatttat aagtgagaac  25741 acgcagtgtg tggttttctc ttcatgtgtt agttatctga ggataatggt ttccagctcc  25801 atccgtgtcc ctgcaaagga catgagataa ttccttttta tggctgcata gtattccatg  25861 gtatatatgt accacatttt ctttatctag tctatcattg atgggcattt gggttgattc  25921 catgtctttg ctcttgtgaa tagtgctgtg atgaactaat cttttcaaat aatcctcctc  25981 tcgtctatta ggtttttttt ttttttggta ccttcttcct cattttatta tttatctgga  26041 taggatggta gcattatgag acgtataata tattaaaaat tatctttata attgaccaag  26101 gcttctccta aagcacacct cattctgttg gtaatatttc aaaatatgga cttagagttg  26161 gtcaaactgt taagtagata atatatataa tgtttttata tatttttcaa tttttttcaa  26221 gctgagacta ttttcttaaa tcaagattta gaggaaaaga atgagaatca agagaatgat  26281 gatgaggggg aagaagataa attgttttgg ctggaagcct gttacaaagc attaacgtgg  26341 catagaaaga acaagcacgt gcaggtagga ctctcataaa tattagagtt attcaaaatt  26401 atgttttcca gtcatttata ttttgacaga tttctttttt ctcccctaat ccaggaggcc  26461 gcatgctggg cactaaataa tctccttatg taccaaaaca gtttacatga gaagattgga  26521 gatgaagatg gccagttagt agttttgatt ttatatgata gaaaatttca gttatatttt  26581 aaatcaatac ctataaaata ccttaaccgt aacttttatt gttagaaata tttttgatat  26641 aggcatttag ttttagatgt tgctgcaaaa tagtagtagg tatgtagtat tttgatctca  26701 tcaccttcag gagttagaaa aggtagaatg agagttatta ttgagagatt tggaatcaag  26761 ggtcatttgg taattcatga atcatggaga aagaatcttt ctattttctg gctgatcgtt  26821 ttaaaatgcc atattaattc atcttgggtg atagaaattg cagagccatc tgtgatcttt  26881 tcttctatgg tgactagcca gcaggttgtc aatatcagaa attagatttg gttagagagc  26941 ttcctatgat gagttcccaa ctgatgtgac agagttgacc tgtcttcttt cgagagggtt  27001 atttgaagct gtcatctctg gataactctt tcaataggag tgccattcaa acatcataag  27061 accggcactc tctcccaaag atacaagctg tagcaaggag ttttgtgcat atcaggtttg  27121 tttcatatcc gtgagccttt gtgttttatg gcaactgatt gattatactt gtgctatttg  27181 caatgggtat cctctgggtt ttaaatagtg aatgacttat tttggaaaca aatagtagta  27241 ttctatgtct gaaatcttga ccgtctattt gtttaattat ctattgctga taagaaggga  27301 attaataaca cagatgctac ttaattaaat attttcattt tgacaagaaa cagtaattct  27361 tttgaaaact atgctaaatt ggcatcttaa taatctcatg ttgagcaagg cttttggaga  27421 ttagggtaag ggagattcat gtcgcagttt ataaatttca gttcatagga tacctatttt  27481 tcaatatcca taagataact ttaaaataaa tatattatta aaacaaagaa aatatattta  27541 cgttatacat ctttaaaacc taccttgctt ctttacaatt ttcagttttt cctctcttgt  27601 ttccattctc ttctcctcac tttacctttt tcctcagcat ctctttacat atctgctgag  27661 cttttttatt tctctctgct gcatatgctt ttgaaaaagc ataaaaatac taatttgtta  27721 ggatttaatt aattcgagtc ttaaaaaatg aactataatt cactcttgta agtggaggtg  27781 gcatgaaata ttgtttatat gctcttaatt gttgttagag atatttgata atggcaagtg  27841 agaatttgag atagttattt aaaagattac tactaacatt ttgtttgaat ttttgaaagt  27901 ttcccagctc atagggaagt gatgctctcc atgctgatgc attcttcatc aaaggaagtt  27961 ttccaggcat ctgcgaatgc attgtcaact ctcttagaac aaaatggtaa gcagtgggcc  28021 atgttttcaa ataaagggaa acacattttt gtggtatttt taattataga agctatatac  28081 tgtgaaaaat ttacataatt tataaagcta tatattgtga aagatatctc tatgtgtaga  28141 gatgtattga catatggatt atgaatatat aggtaaaagg atgaagaata aaataaacat  28201 ttgttgtata tttttccgga cttctatgtg taaactcaca catgacatat acaaaatttt  28261 atgtattttg tagaaatggg atcatattat actcttttat aaccaaattt tatttattct  28321 cttttttatg ttgatacatg tgtattctca cattatcatt ttatttttaa ttttttaaat  28381 taatttttgt ttttagatat ggtctcactc tgtcacctag gctggagtgc agtaccatga  28441 tcatggctca ctacaacctc aaacttttgg actcaggtga tgctcccacc tcagcttcct  28501 gagtatctgg gactacaggc atgcactgcc atacctggct aattttttgc agagatggtg  28561 ttttgccata ttgcccaggc tggtctcgaa ttcctgggct caagcaatcc atctgccttg  28621 gcctcccaag tgctgggatt acaggcatga accactgtgc ccggcccaaa ttataatttt  28681 taatagcttt gtagtttttc agtgcttgcc tgtattctgt tttatgaacc aatttcttac  28741 tgatgaagtt ttagtttgtg tccagtggga tgtttctgga acccatagta aacaccagtg  28801 atcactttcc tcctctgttt tcctttatac atggttccta ttattttctt gggaaaattt  28861 catagaaatg caactgggta gagagctatt caccatttta aaatctggta aaattgtcta  28921 ttataaattt ccacactata cttttttaaa aaatcgttct ttaatgtaat tcttataaat  28981 cttatagctt tgtataatta tgaagagaaa aatggcttgt atccctttag aaagacatga  29041 gttttaagat tatggttcag gctgctcaaa tttcttcccc cataaacagg aatactgcca  29101 gaaatcctta ggtgaaaact catcataaag gcattgggac ttggcagctt ttgcaggaca  29161 tttttagagg gcaaaaaata gagaaaaaca ctgaaagtca gagacagaga ccagtgtaag  29221 catcagcttt taatagagaa actgggtagg gtggaaaaaa aataaagcaa ccacctcatg  29281 catgtttctt tatattatta ttgaagtcaa ataaagagga aaatcatttc ttcttcctct  29341 tcctcctttt tcatctcacc tctccaatgg cactttaata aaacgcttgg agtggccagg  29401 gcactgacag acagacaggg gctgctctca aggataatga gtcaaagggg aaggagaggg  29461 aatcgctgtt ctcgaatctc tcttattcta ctgtgcagtt aaagaggtct ggacagggat  29521 ttcactcctg aaaatgagga ctggactttg tggcttctgt tggggcacct ttagagtgga  29581 ggtagacttt tactatgtac agacaacatt gtgttggtga catcattcat aaccacctgg  29641 aaatctcctt tgatatgcaa atcaaacaac cataactttg tgaaatttcg actgcttcct  29701 attgtggtgt ctgaggactg gttacattca gagtccaccc tgatgtcttt gttcagtttt  29761 ctgctctttc tagttcctta cttctttgtt cctgatcacc tgtcaagtaa aatgtcctca  29821 gatccttttg tattgtcttt ggagttctgc cttaataaag catgaagaac ttgagtagct  29881 cgttcatcaa ctttcttggg caatttctca ttgaaagaca cttgggtgtc tttgggtgtg  29941 cagagctgag catggcttta tgtttttaga aaaaatggct acattggcag gcagaagaac  30001 tgcgtccttg gaatcatgga ggtcccaagg ttgcatacat tttgtgtgac atttttccta  30061 ttcaattaat taactaatat ttattgagct cccaagtgtg tagtgtctgt aggcacttgg  30121 gatgcattca ttaagtaaaa atcccaggct catggagttt aaactgtagt agggaagata  30181 ataagattaa ctaaaatatg taatatttga ggcagttaag aataaaaaat gaagcaagga  30241 aggagaatat gatatgttag acatcagagg agatgaagtt gtgaataggc agccaggaaa  30301 gaaggtgact attgagtaag acctgcaggg cgtcgcatat tgttttttgc ctctgaaagc  30361 agttaatttc ctgttaaaat ggagtggatg agatcaagag tattacgtag atagctggta  30421 aatgcgatag tgtgtaaaat gttctataaa gtctaacgtg actttatgat gaaatttctt  30481 cttctaggtt tattgcttgc aattttcaaa ccacacattg ggttactgtc taggatagtg  30541 attcttaaag tgtggttcct ggaccagcag catttgctgg gggaacttga taaacagtgt  30601 aaattcaagg accccataca gaccttctca atcacaaacc ctggagttga gacccagcaa  30661 tccatgtttt aacaagctct ccaggtgatt ctgatgcaca ctaaagtttg agaaccacta  30721 acccagtgtc atttttgtct tttaaagtgt cttcttggct agaagctagc cactttggga  30781 aaggttatta caacttctga tgtgatcaag caaagtaacc aactctttat tgtatcttaa  30841 tatgtgatat tctgaatgtg tttaaaaggt atgagttttt caggctctgg cagtatttta  30901 gaatgtgtat gtgatttcta tttatttcca tgttttgtcc tatcttctta agatagacta  30961 cttattttaa aagcagtact taagttaaaa ctttttatgt ttctttttct gccactttca  31021 aagtgttgaa tcacagtgtg taatgttgga actgatattt ttatagcggc ttcaagacaa  31081 ttgatattta tgtggaaact tgaagacagt aggtttatgt ttagtgaagg aagtttatta  31141 caaagaggaa aattggccag ttgtggtggc tcacgcctgt aatcccagca ctttgggagg  31201 ccaaggcagg aggattgctt gagctcagga gttcaagacc aggctgggca acatagtgag  31261 atcccctctc tacaaaatat taacaaaaat tagccaggca tggtggcaca cttgtagtcc  31321 ctggtacttg gaggctgaga caggaggatc acttgaggcc aggaggttaa gactgcagtg  31381 agctatgatc atgctactgc actccagcct tggcaacaga gagagatgct gtcacaaaaa  31441 gaaacaccaa caaaaaaaga ggaaaattat tccttaatca ttattgctgg aatatagtta  31501 ctttccacaa atagtgaagt gccagttgta aagcatatct atatgtttcc tagactttgg  31561 cattactttg tgaaaataac tgtaattact tatgttctat gtaaatgctt tccattcatt  31621 tgtatatgat ggcatatata gaaattataa tgtttgtaaa gtccactggg ataaatggac  31681 aaagcagctg aaggctgaaa gcaaccaagc cttttacagc cccttcattc cccacactcc  31741 caaaaagctg agtgaatggt cgatacctcc acatgcttat aactcattcc cagcccacca  31801 gtgtctagca tatctggtta gtcttagctt tatataagtg cagttatttg tgaacttgtt  31861 ttaagtattg gaatacaatt taactttcat tcttattttg gagaccatta tttaaacaga  31921 tttctttttt cctgcaaaaa cactcttttc acaatggaca gagacacggt gattacatta  31981 aaaccatcta ctctatgaat aaaaatgtta aaaccaaaat cccaacaaag ggttaataaa  32041 ggcaaaaaaa attggaaatg acatgtgttt taaagaaata aacatgaatt atctttaagc  32101 tgtcaatgaa ctataaatta tgtgtgctct tgtatatgct ttcctgtaaa tttggactat  32161 attaatattc taaagcttat ggtaaaatta tgaaaatatg ctttcatatc tataagtaac  32221 attttaaaaa atctcagtta atttcagaaa aatactgtta tcaaaaggaa tacacctgaa  32281 tgttttggag ttaatgcaga agcatataca ttctcctgaa gtggctgaaa gtggctgtaa  32341 aatgctaaat catctttttg aaggaaggta atatagattc attaacttgt acagaatata  32401 tcatattggg ccaggtagaa tatcaatatt tcaagcatat ttctaacaat gaaaagaaaa  32461 agaaaaacat aagacacttg aaaactgaag cattttgcaa tgtaatctcg tgtcactagt  32521 accatagact tactttatct gaacactgaa aggaatggca agattgtgga aacatgttga  32581 aggtttgctt ttgaacctga tgcttgatgt tgactatatt ttgaaaagtg gtaattgtat  32641 agcacatagc atacagcagt ttttctaatt attgtgtgtg tgaaagttat aaaagataaa  32701 atcagtttat ggctaaattt tgctctttca caacgaatat attattcctt catctgaatg  32761 aactttgtct tctcttcctg ctctcaatcc ttagttaggg aaaattttaa ctacatctag  32821 tccaagtgca gagatgctaa gattatatag ctggcggtta gtggcacaac agagaccata  32881 tcctttgatc tatgtgtgga tggtggtggg gcagggtggg atgaggtggg ggtgaggggt  32941 ggtaggtgtg ggcagaactc tcatgtgtaa aaaaaataat tggcacagaa gttgcagtga  33001 aaactaattt tgttcctggt tttgccacta atttgagcca actgtttcat ctctaaaact  33061 tcacgttcct cattgataaa gaggaatgat aataacaact ttgaaagttg taagcttaga  33121 atgtaaggat taaataaatt aatttttaca aagggattag gataatgcct gctgcatttt  33181 aagcactcaa caaattgtgt ctattgttgt tatactgtta ctaagtgtga ataaatgaag  33241 tgcatatagc atgaaatgta gcgtaactgc agacttgtaa gaagtagggt tacactgttt  33301 ttaacatcag tctaactaat ctatgtttat atatctttct aagctgtatt tctcttattt  33361 aagtgttgtt tttgaacact gagatgaaaa gtttatctta aatgttgatt ttaatggggc  33421 cggaagtgtg caaaccttta caaatgaggc aaaaacacag cggaataaac tccagtctag  33481 gattctgtag attctgggca aggcatttaa tgtttctcgc tgcatgctct tacgtaaaat  33541 gtgtacagtt gcagccctgg agattccgca ttggctctga cagtgtgtct gcccctacag  33601 aactcatgtg agtcgagaga ctgataagta aacagattat tataatacag tctcagaatg  33661 cagtggcagt agtgtgtaaa agacgcaatg gtaagagtag agtggactca gctggggtta  33721 cccaaggagg ggaggctcca atggagggat gtgtttaaac tgggacttta agttggaaaa  33781 gaaggaatgt atctcagtgt ccacagaacc atgcaaagtg agaacatggt tctgatatgc  33841 acaagtttca gttaacaagc aaagcaagga ttgactgtat taaagttcat agtacctact  33901 gcattctagt caagtgacat ttgctcatat gtaaaagaaa gaatagctta aatacctgag  33961 agaaaccaag actgtaaaac aaattaaaaa taattaaaaa taccttataa gaagtccagt  34021 gatgttaatc tggaagagga aggttcgtgt gatgtaaaga ggccttgtct tggggtcaga  34081 caagtttggg taccagcctt gtctctgtca ctttctagtg ataagacctg aatatttaac  34141 ctctatctgt ctaagttctt catgtagaaa atggggataa taacacctac ctgctgggat  34201 tgttgttatt gacccatcgt aggtcagaag gatgttgtta gttttatgaa gtgaaataat  34261 tcccggatta ctatgaattc tatcttatga gttcaaagtt tagacaatta aaattatgta  34321 tgctcatact actgatttca aatgcatttt catatagtct ttcctgataa aatatattgg  34381 ttctgccctc ctgtacttat ttcaatttgg tgtttatacc attgaatcag atcagtcttt  34441 caataagcat gccaatttta tatccccagc aacacttccc tggatataat ggcagcagtg  34501 gtccccaaaa tactaacagt tatgaaacgt catgagacat cattaccagt gcagctggag  34561 gcgcttcgag ctattttaca ttttatagtg cctggtaagt tacatagttg attgtgggaa  34621 gagataacaa tttaaatgga tttttgattt ttcatgaaat agcaatattc taggcaaata  34681 ttaaaagact agtttctgtc gactaaatgt aaatctttct gttaaaccaa aaagaggtta  34741 aatatgatgc agaagagtca cttagattaa tttttataag aaagcaatat gaattcagta  34801 atttatttat acaaagtaac tacaatgtaa aatgtggagc ttttattttt aaggagggtg  34861 ttcatctctg ataattcttt tctatttttg ttgccatgac ctgagttcaa gctttttttc  34921 tcttgtttga attgtactat tagctaattt tcatacctgt tctcttcctt catttctatc  34981 cttttcttac tctatgattg aattaatctt tctccaatgt ggcttgtact catttacctc  35041 aatggcttcc accctccacc cacctttaat tgttactgac atctgttatc accttatttg  35101 ttctccaaag cccctttaaa acacctatgt ctgtattcat ccaacacatt tattaagtgc  35161 ttcttatgtt ctaagcactg tgatgctgtt aaactttaaa agatgaattg gaaaaaaagc  35221 acaactgtac tattatagga gcttagaggt agatggaagt gtaaatagat aattaacagg  35281 cagcgtgaga gaggcgatga tagatgcata tataaaaggc tttgagaata tcgatggagg  35341 cacaaataat ttcttcaaag gagtaggaca gagattgtaa tatttgaggt ggaagggtag  35401 atagagctgg cctggtagaa aggccaagga agacatttca agcagaagaa agtgcacatg  35461 gtcgggggtg agtgaggcat ggaggataag gagaggagtg cttggggatg agtgtggaag  35521 gaaaggctga ggcccaaaga aaagggcctt gaatgcagtg ctaaaaattt tttgcctttt  35581 aaaaatggaa gccaacccaa gtttattaaa attgtttgca gattgtagtg tcacaccgaa  35641 atttgcaact aaagacaata gcattgtggt gcagaggatg atagacaggg agagagacta  35701 gatacaagga gattgggttg gaggtccatg gtagtcaagt gagacccagt gaagggaccc  35761 agcaatggaa acacagatga gagggcagat tggacagatg gttgggagat ctgtttgatg  35821 tgacatcgtg atccctaagt atgagggatg atactttgtt ctgtgaagtt cccaatcttt  35881 tgaagtcatt tttgctttgt tcttttaaaa tgaaatcttc ctaaatctat cattttctct  35941 agttatatag gtattttgtt tctctgtata agagtattac tcaatataaa agttttttca  36001 aggacagagc tttcttcagt catttttttc tgggcccccc agtcctctgg tggctattca  36061 atcagtattt taaaaattga atcatggtgt cctattcata gtttcagctt agttttgaga  36121 cgtaatgtaa cagatgtaat tttacttgaa aatattattg catgttttat tgaattttat  36181 ttttagattg taattaaaaa caataaaaat gccttttgat tatccttaaa gttgacagcc  36241 ttattctttt gagggaggtt ttgggtttta aagataacca aaggacactc aaaaaccgtt  36301 tctgttagtt aataaaataa tactctttta gtccaaaagc aagttttgaa tacagtattt  36361 cttttctttt attgttctta aactgatcct gaaggaaaat tgttagttaa caatcaatca  36421 gatgtattat gggtgccctt gaaaataatc acttgaggac tgtacttaat gtaaaaaaat  36481 acattttata agcatatcag tatgtaagta cattcttcta gtaggtaaag gcttaatcat  36541 ttactgtatg ctaacatatt atactaccac agtacaccat actgtactgt acccaacgta  36601 atgtaccatg ccgtgcagta ccgtatcatt ctaggtatca acaagtaact atggtagcaa  36661 tactccagta gttttgaggt tgaatatgat tctggtttga acatgttaag ttgaggttct  36721 tatgagatac ctaggtgtac atgtattcat ttattagcta gattatattc aggtttttta  36781 gtggcaaaga tgtacattat ggcaaatgtg tataatttag ggtccaatta aggatagcat  36841 attgcatcta gtgcatatat cttaagtttc tttcaatcta ttactgtact ttttctcttc  36901 tactttataa taacttgtta tatagacatt gagctggttg ctttaagaaa ataataatga  36961 agacccagtt actttaataa aatatcattt gacttgtttc ttgagttact gctagtttta  37021 gaggaaataa tgtgaaattc tttaagaagg gatataggca aattgggaag tatatagagg  37081 agagtgatta gggagataaa agcacatcaa aaagagcaat aagaggaact gagattgttt  37141 tatttgaaga agagatgact caaagggata tgaaactcta ttcaaatcta cttacttata  37201 accagtaaca gtaataatac caaacaatat ggaggactta aaaagtatta ggcattgttt  37261 gaagtatttt aagtatatta tctcttctga ttctaacagc actccatgag gcagatatta  37321 ttattattat tattttggta ttgtatggac tacaaaactg aggcatagaa ttgtagagat  37381 ttttaaggtc aggcctgata taacagcacc agattttcag cttatgcagg ctgactccag  37441 agtaggcatt ttaaaataaa tatttgtgca acaatcttgt cctgaacaac tgttgtattt  37501 aaagcactat gtctagaatc cttaaggtat cgggagatga tgagattatg gatactgctc  37561 tcaaaaaaat ttacagtcca cacgacctaa aggactgtta tgtagaaagg aaattatatc  37621 catttgaata gtaggacctc aagtgataga acaaagactg gagaagaaat ttacaggaat  37681 gttcatttgg tcttaattta aggaagatgt taaataagac ccattacatg tagcatgatg  37741 tttttactac tcaggccagt tttagtggtc acatcttctt aaggtgtaat aggcagcctc  37801 agaagttact gtggtcttgc tcactggaaa tgatacacag acagtttaag ggacttgccc  37861 cagaccacac ggcaagaggt gaatgtcaga acctctttga gtacatttta aaataaggac  37921 tgaaagttgg aggagggtgg ttatcaaggc tgccttcctt accatagtat tcccagcatt  37981 aacaaaatcc ttggcatgta attggaattc agatgcttct taaataaatg aaaagcctgt  38041 tgtagccagc ttacagtttg cattaatgca gattattaaa gtggaagatc ataaatgatt  38101 ttttattaat atttatgtct ataatcttag gtttggaaaa cattattcat tcataataat  38161 tttaattata tgttacatta ccacattttt gacttgtagt gtttttagca tagttcagct  38221 acagtgtagc ttaataaaga atatgatttt ttaaaatagc aatgctatta tatagccttt  38281 acagaacttc taaaaaatga catgttctct accaccttaa tactgaaact caaatcttat  38341 tttttgctac gattattcca gctactcttt tttgtctata tttcatttct gcctttttat  38401 gttgtggtcc aagtaactct gagcttttct catgttgtcc attgttgcat aaaaatcttc  38461 cagcatctta aagcacagcc tactcacaca aaaaagtgat tgtttgctac agaaaatttc  38521 ttcaccatcg taattttttg ctacttcaaa ttcagtaagc attcttacac attatattta  38581 ttttatattc agtgatgaac tatttttata gattccttaa aatttctggt tatttatttg  38641 ataaggaaac atgtactaga aaaaagtaca acacatatat tgtgagatta attatgacaa  38701 tttctagaaa gtaacagtct gttcaactca aatgtttata agaaaattct ttctttattt  38761 atttatctgt gcatttaggc atgccagaag aatccaggga ggatacagaa tttcatcata  38821 agctaaatat ggttaaaaaa cagtgtttca agaatgatat tcacaaactg gtcctagcag  38881 ctttgaacag ggtatgttga atataagttt tctgtattta tactattaac taaaatatta  38941 aatttggaga actaggggcg ctttttcagt ctaagttttc tgttctccgt ttgctatgat  39001 aggaggaagt catgtggtta gagacataag atgacagtgg ggatgtggga agtgaaaaga  39061 tatgtactaa gctaagtcca gctaagtgta ttatcaatta tagatgtagg caagattctt  39121 ttgattgcca gtaacataaa tccactctag tttgctcaac cagaaagaga accaaagagc  39181 catatatgca gctagacctt gtgagtcatg ctgggtacta tggctgctgt tttctctttc  39241 tgtcctctgg ctacttgtct ttcttttctg gtctcatagt atatggttta gcccatgaag  39301 acataccagt gttaacagta aagtcttcgg ctgggcacag tggcccacac ctgtaatccc  39361 agcactttgg gaggctgagg tgggtggatc acgaggtcag gagttcgaga gcaacctggc  39421 caacatggtg aaaccctgtc tctactaaaa atacaaaaat taactgggca tggtggcacg  39481 tgcctgtaat cccagctact caggaggctg aggcaggaga atcgcttgaa cccaggaggc  39541 agaggttgca gtgagctgag atcacaccac tgcactccag cttgggcgac aagagtgaga  39601 cttcgtctca aaacaaaaac aaaaacagac aaacaaaaac agtaaagtct tctttgattc  39661 cctacgctcc ttttcattgt tctccggaga aataacctct gaaatgattt ggtatacatt  39721 gtttccattt tttagcattt acatatccat gttcctacat tataattaaa gtatccataa  39781 atcatactga gtatgaaaaa gagaagaagg gaattacatt taaattgtgt aatgcaaaaa  39841 gtattggtgg aattaagaag ttttggaaat tttgcataag atgaattggt tctttattaa  39901 agatgttaag aataaagaca taattagtgt gaacattttt ataaaaggag gagcctattt  39961 aaaataatta atggaaatga ttccatgtga tttgatatac tttgatgaat gtcataaatt  40021 aattaaagtg gcttccagag agatctccct taaaaattca ttttaaattg aactttatac  40081 tgtcactcac tgcctataat atgtttgagt catttatact caaactttaa tacaatcctt  40141 gagtatggca agaatttatg ttgtaatggg ttaaatttat cttgagaaat atttgttgaa  40201 aataagtata tggaaggaag gggttaggca tttagaagat aaataaatat gctttgtact  40261 cttctctcct gaatctcata agccggttgt tgatggctgt tgtgaaacct tggttctttt  40321 ctttaaacaa gagacacaca gcagaggaga tgcagcatcg agtaatttat tgcaaaagaa  40381 aaagaatatt ttgcaagtta agtgaggaat agacacttat accctgacag aattcagggt  40441 gggcttacta gtaaggatga gacagcgtaa attggcacta ggaagactcc ctttgtggga  40501 gttgtacatg atttttcata agtgggtggg aagaagtgtt actagtaagc atattctagg  40561 ttgtcctctg agtgaacatg tgcagtagct gtacatgctt gttcatatat cgcatgtctc  40621 ataagtatct gaaatctcca cccaggggtg tgtgttttac tattataatg agcaaagggt  40681 cagtctgagg acaaggaaaa tcaaaatgtg catgctcccc acgctacctg acttcaaact  40741 atactacaaa gctacagtaa ccaaaacagc atggtactgg taccaaaaaa gagatatagg  40801 ccaatggaac agaacagagc cctcagaaat aatgccgcat atctacaacc atctgatctt  40861 tgacaaacct gacaaaaaca agaaatgggg aaacgattcc ctatttaata aatggtgctg  40921 ggaaaactgg ctagccatat gtagaaagct gaaactggat cccttcctta caccttatac  40981 aaaaataaat tcaagatggt ttaaagactt aaatgttaga cctaaaacca taaaaaccct  41041 agaagaaaac ctaagcaata ctattcagga cataggcata ggcaaggcct tcatgtctaa  41101 aacaccaaaa gcagtggcaa caaaagccaa aattgacaaa tgggatctaa ttaaactaaa  41161 gagcttctgc acagcaaaag aaactaccat cagagtgaac aggcaaccta cagaatggga  41221 gaaaattttt gcaatctact catctgacaa agggctaata tctagaatct acaatgaact  41281 ccaacaaatt tacaagaaag aaaaaacaac cccatcaaaa agtgggcaaa ggatatgagc  41341 agacacttct caaaagaaga catttatgca gccaacaggc acatgaaaaa atcctcatca  41401 tcattggcca tcagagaaat gcaaatcaaa accacaatga tataccatct cacaccagtt  41461 agaatggcga tcattaaaaa gtcaggaaac aacagatgct ggagaggatg tggagaaata  41521 ggaacacttt tacactgttg gtgggactgt aaactagttc aaccattgtg gaagacagtg  41581 tggcgattcc tcagggatct agaactagaa ataccatttg acccagccat cctgttagtg  41641 ggtatatacc caaaggatta taaatcatgc tgctataaag acacttgcac acctatgttt  41701 attgtggcac tattcacaat agcaaagact tggaaccaac ccaaatgtcc aacaatgata  41761 gactggatta agaaaatgtg gcacatatac accatggaat actaagcagc cataaaaaat  41821 gatgagttca tgtcctttgt agggacatgg atggtactca gcaaagtatg ccaaggacaa  41881 aaaaccaaac accatatgtt ctcactcata agtgggaatt gaacaatgag aacacatgga  41941 cacaggaagg ggaacatcac actctggggc ctgttgtggg gtggggggag gggggatagc  42001 atttggagat atacctaatg ttaaatgaca agttactggg tgtagcacac caacatggca  42061 catgtataca tatgtaacta acctgcacgt tgtgcacatc taccctaaaa cttaaagtat  42121 aattaaaaaa aaatgtgcat gctccataca ggggcaattc cctactggag atagctttgc  42181 ttaaatgagc tggactacaa tgcaaatgct gaaacttact atattgacag taagattgcc  42241 acagttgccg cgtcctgagg acatggttac ttccttttaa tacctatcct gtctcattgt  42301 gagaggatta acaactgtgc ataaaaccag ttgttctaca tgagcactta ggagggatac  42361 cagcattgtg aacatagttt aagtacgtag aggagggaac agttaagttt attcatggtg  42421 agtgttggtg aaaagtggaa gaggtaccaa aacagccgta tagataactg gttccagtta  42481 gccaacattc tctaaagtta ttagagaagc ctaagtgagg tgtaacctca gcagtcggga  42541 gccaagagag caagtaagtg ctgtgatgtg gagaaaatca ctttgttcca actgagaaga  42601 aatggttgag cactgctttt cccccatgcc agtactgacg cacagccttt cacttagcac  42661 tgattatcga taggggtggg gagttaaggt atggggaaac acaagtaaca atattttatt  42721 tcaaaaacct ctccactgta attcccctaa tccttcatca tggttgagga aaatggctct  42781 aaaaaatgag agcaattact gtagctccaa aattctgtga ttgcatgtct tactctgaat  42841 agcaattaca aagcatcaga ggatttaggt ccaaatattg cagacacaag aaaatgaatt  42901 acattttaat acatctaaac ttggagagca gagttccaaa taaggtagaa cttgagattc  42961 aactctgatt tataaagcag agactaagaa gagtatttat aaagcgaatc catgtttgga  43021 tacataaaag tgcaataaaa ttcaagctga agttaaaatc tctgtctaga acagcgatgt  43081 tccatttatg cctgatcctt ttagcttttc cacagatgaa gactttgtca cctgttccag  43141 agagatatat ttgttcatta ttgtttccag agagcaaaat ggaaaataaa ctctgcacat  43201 tttggccgca tctgtgtttt atatgcggtg acactcctgt tctcttcagt gaggaaatcc  43261 agtaaagtaa aaccagtctt ctgatgaaat gggcacaaat caaagaactt gtgagcttca  43321 caaaaacctt gaagcaaaat ataccaagct taaatattga atgtattgat ttcagtagtc  43381 aaaaacagag ctcatctgca aaagcaacaa caacaatgac aacaacaaat tacatataag  43441 taaaatttaa aaaaggttta caggatgaat atacagaaaa ctatgaagct taggggtaga  43501 gaggagtagt tgaatatatg aaaagataca tcttttttga tgaaggacta aatttttaaa  43561 aatgaaaatt gatctatgtt tatgaaccat tagtaaaaat aacaatagta ttttctggaa  43621 ctaagtaagc tagtgtaaaa tttgtatgaa aaaattaata catatgagta atcaggagaa  43681 ttttaaaaaa gagtgttgat tcatatggcc taactccaaa agataatcaa atgtattata  43741 caattttagt aattataatg gtgtgatact ggcactaggg gagagagatc agtgccacag  43801 aagggtggct caggaaatag actcaaataa aaatttgtaa tgttatgata ttcctcgact  43861 gcgggaggaa aatagattac tcagttactt gtgttggaac aactaactct tcagccattt  43921 gggaaaagca aagaaagctg aatatttatc ttactccttt tgccaaaaca aattacagat  43981 ggatgagatt taaagtctat aatgaaattg gtatatgtac atattaaaag tttcaactat  44041 aattgttatt aacgtaaatg acaacggaac accttgttgg agggaaattt ggtaatatct  44101 atcaaaatta aaatgccatg ttttctgatc agcaatttca ttctaagaat ttttaatgta  44161 gatatacttg ctcatgtaca taaagattat tagacatgaa tgttcactgt ggcatgattc  44221 gtaattaaaa atgtggcaac aaactaaatg cttacatggt aatcattcat gctgtttgcc  44281 agatattttt atttctccac cttgtggtga ttctggcata gtatttagta gttaggttga  44341 agcatgtgac taattttggt cagggagttg taaatggagg gaatagctgg tgagacatct  44401 tgcggaatac tgtctcaggt accatcgtac ctgcagtgtt tgagatagta gttgctccat  44461 cagtagaggt ccctgaggga ctacaacaag cagagtcccc tcctgacctg caacacatat  44521 gtagcatgag caataatgga acttattatt tttgcaccct ataaaatttt gagaattgtt  44581 actgcagctt aatttaatct ttcctgacaa tacaatacct atcaatagag actaatcaga  44641 taaattatac catttccaaa ttgtatcatc ctgcataaat attaaaaaac aatgaggtga  44701 gattcctaat gtgctgctat ggaaaaatct tcaattttct atgtgtacga atgtatttcc  44761 caggtattcg ttttttcctt cctgtatgtt gacacattat gcacactttt ggatgaaatt  44821 aatatatttc caccgcttta tgtcctctcc ctcactttta tcacccaatt gtagcaaagt  44881 atatgtttgc ttttatagct ttagctatat aaaattttct aatagctaag tttgtggttc  44941 ctggaattaa gatatctgaa tttaaatcta acactactac ctacagacta tgcaccctgg  45001 acaagtactt aatgtcttgg ttttgttatg tataaaatgg agataataac tgatttttca  45061 cttagagttg ttgaatattt tataagataa ttcatgaaaa agtgtcagta taatgcttgg  45121 cacatagtat gcgctcaata aatgttaatg ttattactat tagatttaaa agtatctttt  45181 gacccctggc tatagaagat gaggaaatca gagtatttgc acttctaata tctcctgttt  45241 tcccacctac ttttgttcaa taaattaact ccacattggc agggtagata atatttatat  45301 tcagctttct aattatgctt tctaagtttg tgtttatctt actcctaccg ttatttggaa  45361 gcagtcttca tctcaagtcc ttttgttact gttttttcac tcatctttta gttgtctgaa  45421 tttctttaga aaagtttaat tccctgaata tttaatgttt atattttgtt gttgtttttg  45481 cctttatatt tggactccta aaaaaagttt tgttggttgt aaaatattgt ttttttttcc  45541 ttgaagtgtt tgtaggcatt tcactgtgtt ctagtactga atgttttagt ggagaagtct  45601 gaggccagct tcatttgttt cttggatatt tttgcccagt gctgaaagga ttttttctct  45661 tgaaatccat ccactttatt agggtatatc tcaatgttag tcctgggata tggtatcctc  45721 attctagcta tacattcaac ttttttcctg gaaagttttc ttgagttgca tctttatata  45781 catatacata cacgaataca tatacatggt aaagatatta aggataaaga taggtgcttt  45841 ataaatacca atttgatata attttggcat gaaaaaaagc ctgtggtgca ttctgagttt  45901 ggaagtagaa ctggatatac ttacttatta ctattgtttt taatacaaaa atggagacag  45961 ggtctcacta tgttgcccag gctggtctca aactccttga ctcaagcaat cctgcctcga  46021 cctcccaaag tgctgagatt acaaatgtga gccaccgtga ccagcctaaa tatctaaatt  46081 tttctctttc attattttag ctctcttctt tggggctatc agtcatatga tgttggatct  46141 cttttgcctt aatcttctaa tttttcaaag actaatccag agaccactat ctatcaccat  46201 gccattactc atagacaagg gacatcattc cacacattta gcttatttgt caaaatcaga  46261 gatgggattt ataaaagaaa aagaataaag ggagaaaaca aaacacaaat tatctgggaa  46321 tgcgagtctg ttttcctttc tttggctaat ctacttacct agtagctagg aaagaggcca  46381 gcatgagtat ttttccattg agctggctag ctcagtcatt ggagaaacca tttcaatatt  46441 tatggattct ctgaaaaaaa cttggagagt aaaaaagcat aggtggtagg atgctgccat  46501 ccttctgttg gcatctccag agtttcactt gaaaaacacc tcctaagtag acactgataa  46561 attgatttta atccctattt gagcaccaat gcaatacatt actatttcaa gatggagcat  46621 tagataacta aaggaaattc tattgtgggc ttgtaaatat catgatagtc atacctatgt  46681 aatacatata tgtatatata aatattaatt tttaaacatt tacttagcta ttaaaaatca  46741 aatgctcata tttaaaatta ttggtttatt aatgcaccat cacatgttat catgtgatac  46801 tctgaattct ctttttgcat agacaaatca gagattacct gctgcaagtc attagtcatc  46861 attaacctgt actgaaatgg ttagagccta ggggagttcc aggtaaaagg accagtaatg  46921 agtagaagct tgttagatgt agagatattg aggacagaga taggtgcttt ataaatatca  46981 atttgatata attttagcat aaaaataagc ctatgatgct ctttgagctt gggagtagaa  47041 gtggatatgt aattttcagg gcgtagtata aaatggaaat gcacactcct tgtttgaaaa  47101 ttattaacga ttttacaagg gtgacaacag agcattgaag caagtgctct gtgagcatga  47161 agccctgtgt gaaatacaca cctgtgaagc tggccttgcc tgccaaacag gaatgctggt  47221 actgttaatc aaacagaaag ttcagaatga caatctgact tttttttgta gaagatacta  47281 aacttttggc cttgaatatc tgaatttaag atactggtat caagccaaaa gaaatttggg  47341 cttagaactt gaaagattta aacttgtgct ttgacttgtg tgctcacagc tctctgtgtc  47401 acaatttttt tcatctgtac ttcgggacaa caatagtgtg tcgacatcac aaaggttttg  47461 ggaggattaa gtaggtgaat acatatgaag ttcttaaaag aatgtctggc actgagtgag  47521 tgctacctcg gttttggcat cgttattgtg gtcattgcta ttgttattat gacttgaagt  47581 catattagtg tatgaaatcc catccatgaa tagaagacag aaagaacttt gggcagggtt  47641 tggaggtaaa agaagacatt gtaaaggaga tgggtaaagg aatgatttta aagactgaga  47701 ataattaggg gagtacgatg tcatggaaat taagggatga taagcaccaa ttaagttgtt  47761 attggacttt tttctttgaa tggttcaaat tcagaataat aaggaaagaa gtctgattat  47821 acgaaattaa agggtagggt gactgtggag gtagtgggag ttgacttttc tgctaataag  47881 tttagaaata aaggaaaaat ggtagcttga ggaaagagag gagcgattaa gggaaagtct  47941 ctcgttaact cttgtttttc atctctgagg acagcgctta gcccaaggcc tgacctgtga  48001 tcattactct ctcaaggttt attccatgga cagagctatc tcatttcatg cttataacaa  48061 ccctacatta ttagaattgt tttagagatg aggaagctga gacacacacc aaaccagcct  48121 tccaatttca ctttgcacaa ctttgaattt ctttatattt cttgaataaa agttccactt  48181 tttaacttac cacttcttag cagtcattgt ctaactgagt aattgttact tcattcattt  48241 aatggttctc agattcgcat aatttgaacc taaatttaat tggcctccaa gctgatgtgc  48301 ttacagaaac agtgacagga aacaaaaatg tcaagggaga ctatgtatta ttaagatgat  48361 aaatgaaatg atgtccaagc tgagcaatta aagtgtgaag tagaaggaca cagggtgaga  48421 aactgatgct tctcctcagc ctctataaaa aagatactga ataaagataa ttgagaggca  48481 ttaggggact agactgagaa aggattggaa atctgttcac tgagagtaca gaaatgagga  48541 agcttggaag gcagaagatt ttggtcaaag acgtctggct tgaagctatt tcagctcttt  48601 ggattatctg tggtggaggc catcacgtct ttggagtgga ggtaccatga aactagtgtc  48661 tgcaaaacat catctaaatg aaagcaaaat tcctgagaag gatggcacta taatttttaa  48721 aagaaaagct atgagttaag cattcatatc aaggtagatg tttggagtgt attgcttgtg  48781 tgtgaaaagg cagagatgac cagaataaga gttagaggta tgctgcgttt tcttcttggt  48841 tgatgagtag gatggcctgg acaaagaagt gacctctagt aaaatacctt catagtgtca  48901 aatcatctgg aggaaattca gattaaagag gttggatgat gtcgtaatta agatcctggg  48961 cttttaaggt ggacacattt atattcaagt cccaggccca atgcatatta gctctgttac  49021 ttgagctttt atttctgcat ctttaaagtt tggcaaacct attacatgaa gctgttgagg  49081 ggataaatga aataacgcat gcaaagcact tgcagtaaag actaattatc aatattttat  49141 ttgttaagag gcagcattgc gttttactgg tcaagtatgt agactctggg gtgaaacata  49201 tttggtttgg tttcatctct gcaatttata gtttgtgtag actttgagaa tagttctcaa  49261 tcattctaac cctcagtgaa ttcatcttct aatgggagtg atatcagtat ggatttcatg  49321 agattatgaa aagaaaatgc ctacaaagta tttattacaa tgcctggcac agaacaagct  49381 ctccttaatt gtaaaaatgc taactcttat tcttcataat aaataaaagt aattaatgtt  49441 atagaaaaca aaatcaagga tactgattta tatttggatt acttgattta tattttgtca  49501 gtctataact ggtcttaact aaggtaagta ttaagatctc atttttaaca gcgagtattc  49561 ttttgatttt agttcattgg aaatcctggg attcagaaat gtggattaaa agtaatttct  49621 tctattgtac attttcctga tgcattagag atgttatccc tggaaggtgc tatggattca  49681 gtgcttcaca cactgcagat gtatccagat gaccaaggtc agtacaattt gaattcagga  49741 tttagaatag atttttgtag ggcattagct ggtgactgga tgtctttaaa tatttttctt  49801 cagttttgag atttaaaaca attctttttt tttattttcc tagaaattca gtgtctgggt  49861 ttaagtctta taggatactt gattacaaag aagaatgtgt tcataggaac tggacatctg  49921 ctggcaaaaa ttctggtttc cagcttatac cgatttaagg atgttgctga aatacagact  49981 aaagtatgtg cattatcttg gaaagaattt gggaacttgt gcgaatttca cttttggagc  50041 agtttgtgta attcccactt tgcatgaatg gggtattcta gttaatggaa aaccatttat  50101 ccttttgtag tattttaatt atacaagcaa agaaaattgg attgaatctc taaagatcca  50161 gtgtttcatt atgaaatctc taaagtcagc atggttattc accatttatc ttgcccataa  50221 aagttcagag aatgtgctaa gaaatcccag ctagctgagt ttattcgctt agattttaga  50281 taaatagaat ttataaatat tccaaagttt gtcactctct gggttttatt gcaggttgct  50341 tacctttagt aattttgctt gttgattttt ttccttgcag tgaaaaaatg tttttaacat  50401 ttttcatcaa gcaaaattta aaacatgata tataataact gtctttgtaa ggaattcaag  50461 atactggcct agagttagtt cacgggagat taagaataaa tttgttttgt tttgtttttt  50521 aattgtagca aaacaaatag tttttcttca agagtttctg ccttggttgt ggagtttgca  50581 actttcataa actacaaagg aatttttttt ttttttttgg agacagggtc tcactctgtc  50641 acccaggctg gagtgtagtg gcagatttca gctcactaca acagctgctt cccgagctca  50701 agtgattctc ttgcctcagc ctcctgagta gctcagacta caggcatgca ctcccatgcc  50761 tggctaattt ttgtattttt tgtagagatg aggtttcacc atatttccca ggctggtctc  50821 aaactccctg gtctcaagca atctgtcctg ctcagcctcc caaagtgctg ggattacagg  50881 tgtgagccaa ggtgcccagc tgactcagga aatatttttt gtaactggca gcattgacca  50941 ggaataaaaa tacctggtct ctaatctttg cacagacatt atcagtaaat gagagaatat  51001 gtgtaaagtt ttttaaaaaa ttataaagtt atgaacatac aaaattctta gattaataac  51061 aacaatgtgt tttataactg cttttcataa tgtgcctcag gctaggctga ttaaaccaag  51121 ataggattga ttaaaagtaa tcttagggaa agggaaggat tttgtgccgg tatggaactc  51181 tcagttactc tggattaatt catctaggca taaattttag aatctctata gtagagttta  51241 tgaactaaat ctggcctgcc aacatatttt atttgtccag ttcagggttt tgctttgttt  51301 tttgagacag agtctcactc tgttgcccag gctgtagtgc agtggcgcag tctcagctca  51361 catcaccctc cgcctcctgg gttcaagcaa ttctcctgcc tcagcctccc tagtagctag  51421 gactacaagt atgcaccacc atgctcagct aatgtttgta tttttagctg agatggggtt  51481 tcgccgtgtt ggccaggctg gtctcaaact cctgacctca agtgattcac tcacctcgga  51541 ctcccaaagt gttgggatta caggcatgag ccactgcacc cggccttcag ttcagtgttt  51601 aaaagttttt aattcgaatg acgtactttc tgcacatttg catggcctgc tctgctgtag  51661 cattcacttg ttttcagaga cctctgctct agaggcaggt ggatcacctg tccctcagac  51721 atacataaat taaggctact ttgcttatca aatattagta ttcgtagata ctcagcatca  51781 taagagttcg aagtaataat tttaatattt agatgacgta agttaattta aaattttttt  51841 gagatggggt ctcactctat ggcccaggct agagtgcagt ggcacaatct cggttcactg  51901 caacctctgc ctcctgggcc catcctcctg ggtgggctca agtgatccac ctcagcctcc  51961 tgagtagctg ggactacagg tgcatgcacg ggtaatttta aaatattttt tataggcaca  52021 agattttgcc atattgtgca ggctggtctt gaattcctgg gctcaagcaa tcccacagtg  52081 ctgagattac aggtgtgagc catggtgtct agccaatttt attaatatgt aatattagag  52141 gtaataaaac attaaaaagt taagatgatc cttggtggct ttacccaacc taaataatac  52201 taaagtcaaa agcccaatct ttcattaaaa catcacatga gtgaagagga cagactctgg  52261 ggatgtgctt aaggtggttc taaaaaagta acggtgttct ttataaataa cttattatta  52321 gaatgtaatc ctcagagtgc cctcagcgct tctcaactac actcaacata aatgaaatct  52381 aggagtccac actagccttt ctgagataaa catttcggaa gacagcgcaa aaagctgggg  52441 gatatgctag gctctctaga gaacctactg ttcaatatta taatacaaat ttttactcta  52501 ttgacctgtt tggatgtgta gttctgctga tccaaccgct ttaatcctgt ttaatatctg  52561 ggtttcatcc tataactatg gctttagaca agcatctttg aaaaccaaat ttgagggtat  52621 tagttctttt tcctgctttg ctactgaatg gtttgttaac tagcatttta ttctctgtgc  52681 ctgctatatt tcttagtcat gagagagaga gggagtattt atttacagga taaatacttt  52741 aaagcaccaa cccaatatat ctatagttaa atgaacatcc taggtattgt ttcatataca  52801 aactctctct gctttatact gtttattcat tttgcctgta attgcttatt ttattatttt  52861 ttttcttata cttttaggga tttcagacaa tcttagcaat cctcaaattg tcagcatctt  52921 tttctaagct gctggtgcat cattcatttg acttagtaat attccatcaa atgtcttcca  52981 atatcatgga acaaaaggat caacaggtac agtgtttttc acttgcatcc taaatgttat  53041 gtatttatct gactctaatt ctcatttcca ctctttttag tttctaaacc tctgttgcaa  53101 gtgttttgca aaagtagcta tggatgatta cttaaaaaat gtgatgctag agagagcgtg  53161 tgatcagaat aacagcatca tggttgaatg cttgcttcta ttgggagcag atgccaatca  53221 agcaaaggag ggatcttctt taatttgtca ggtaaatatt caaggcctca cttttgtctt  53281 tgctcagtat tcttatagaa tgtaagagcc ctgccattgt gtatctctta cttatatcat  53341 attattcttc actacagaaa tttaccagtt tattgcaatt gtttgtgtct tgtagtagat  53401 ttatagaatt ccagaagtaa tagggtcctt taggtgttat ccagtctaat ctttcatttc  53461 atctgtttac ttatcttgtt aagttgataa ataacttttc aaatgtgtcc cttagtaggc  53521 atctctacaa cttagtctcc agatacactc cacataacac atagttctaa tgttttgata  53581 attttttaac catttttttc catggtttta gtttctttgc ctagaaagtt ctcccctgag  53641 ggctaccaca catggctatg caggctgtgg atggcacact tttgtcggtg ccattcacag  53701 tgacatgagt tgctgttggc caaagttgtg taacactggt ctttctttcc ttctctcttc  53761 cctcctgaac catgtaaaca tatatctatc tgattgttct gctctccctt caaaatataa  53821 ttcaaattat ctttctttaa agccctcccc atacctccaa acctccaaac aaaattaaga  53881 tttacttctt ttgtcagtct atgaaaatat atacatatct cttgtatact tggtgagttg  53941 tgtgaaaata acagtgtaca gtgttcatct ttgtatcatt cagaatatcg agctcattgc  54001 tttacatatg gtgtgtattc aataaatact aggttcattg cttatatttc agatttgtat  54061 tatttgtata agtgttagag tttatactag cattcaggta gcactatgtc tattttctag  54121 aaatttaata tttctaacaa agcaattatg tagtgattta atacacatta ttaaataatc  54181 aataaagtac tatgtttgcc aatagtttac tttttaaacc ttactgtatt taatatccct  54241 actgtattta atatcccact tgcctatgga ttgaaatcaa tttgttgact gttaagatta  54301 agttaatact aattagtaat caacataaaa agaaaaagaa tttgtaaccc attttcatgc  54361 attacgttta tgaattaaaa tcacataaac aatctaatta tttaaattta gtcaaatttc  54421 ttttaagcaa gcaacaatta aaatagttgc tccgctttac taaagataat taaatttttc  54481 catcaataat ttaatacatt tttactgtgc atcttttgca tgcagattat tgcattaatt  54541 ttaattgaaa ataccgaaga actaaaaaga aacttcccct tctaagtcca cattaaggaa  54601 acaacatacc taaaagcacc tgatacaact gtactacatt ccccacagga aatcatttct  54661 actattcttt caatttatcc aaatctttct acccaacagg atttttactt tattcctctt  54721 tccatattct tttggacttc atatgcttag ttttatcttt tctttttaaa acgaaatctt  54781 aaatccaagg attatgtatt aggtttaaag aatttatccc agttgtcaga ggttatttat  54841 atctagcaaa caataactgc tgattaaatc ttgtggatga gtttgtcgta tgtaccttat  54901 ttgtgccaga gcaaaataag gtaatcagga ctatttattc atttaccaag aggttacata  54961 ttgaaggact atctagagca agggtggagt tgtgttagac tttctgcaga gaatttgata  55021 atggaatgta catgattggt agagaagaat atggaagttt aatactgggt atgcaaatgc  55081 atggataaaa acctcaaggt aaaactcatc aaatcacagt ggaaaaagta tagtgaagtc  55141 tgaataaaaa taataagagg ctgggcatgg tggctcacat ctgtaatccc aacactttgg  55201 gatgttgagg tgggaggatc acttgagcca ggagttcgag accaacttga gaaacatagt  55261 gagactccat ttctacaaaa caaaccaaca agcaaaaaac catgtatgat ggcacacaca  55321 tgtagtccta gcttcatgca gggtggctca tgcctgtaat cccagggctt tgggaagtca  55381 aggcgggagg atcatttgag cccaggagtt caagaccagc ctgggcaaca tagttagacc  55441 cccatgtcta caaaaagtca aaaaattagc tgggtatggt ggtacctgct tatagtccca  55501 gctacttggg agtctgaggt gggaggatga cttgagcctg ggaggttgag gctgcagtta  55561 gctgagattg caccattgca ctccagccta ggcaacagag ccagaccctg ttaaaataaa  55621 ataaaataaa ataaaataaa ataaaataaa ataatataat aaggctgagg tgggaggatc  55681 acttgagcct aggaggtcaa ggctgcagga gctaagattg tgccactgta cagcagcctt  55741 ggtgacagag ggagactctg tctcaaaacc aaccggtcgg gtgcggtggc tcacgcctgt  55801 aatcccagca ctttgggagg ccgaggtggg tggatcatga ggtcaggaga tcgagaccat  55861 cctggctaac ccggtgaaac cttgtttcta ctaaaaaaat acaaaaaatt agccaggcgt  55921 ggtggcaggt gcctgtagtc ccagctactt gggaggctga ggcaggagaa tggcgtgaac  55981 ctggaaggcg gagcttgcag tgagcctaga tcgcgccact gcactgcagc ctgggcgaca  56041 gagtgagact ccgtctcaaa aaaaaaaaaa acaaaaaacg aaccaaccaa ccaaccaaca  56101 aaacaaacaa acaaaaaacc aacaaaacca aacacttcta tcatgctcat taccacctgg  56161 gcactgctcc aaatacttta cacaatttaa tccttacgac aacctacgaa aaggtccagt  56221 aggttctaat gttattccca ttgtgcaagt gagaagctga ggcactgagg gtttaaataa  56281 cttgcctaag aacaagctcc tggtaacagt gtgaaatctg cctccacagt gcctgcttta  56341 atttcttggc tacacagcag attcatggta gtggtggtag tggtgttcat tttctctaaa  56401 ataacagttt gaataatttg gttttgataa tgcactgcat ttattataaa ttagatgatc  56461 agagaaagat tgcagggata agaaattatg cttttgataa tctttagtta tattcttaat  56521 tttcttcatt attatttaaa tgtaaaaata aatatctgtg agcagtagta ttttcctgtc  56581 atgaagctga aattactttc ataaatatgt gtgaatattc taaagagaat gactctgtag  56641 gatttaaaga aattaattct tatttttgct ggcatttatt tattttatca gattcacttt  56701 ctcatatatg tctctcttca tggcaccata tgcctaaagt cagcttggat agtttggatc  56761 ctccaaggaa aattccttcc acaaacatgt gcagcacaca gtgctagata attaatagag  56821 aatataaaat gggtttcctg tttcaagatg gtttgtaggt ctgtatgtgt agggcattga  56881 caagagagta aaacataaat caccttagta caaagtaagg agtgaatggc atatcttaga  56941 gaaaaaaaag ttactgggct ataagagaag gcatttgtga gtttttccct ccctccccgc  57001 ttcccttccc ttcccttccc ttcccttccc ttcccttccc ttcccttccc ttcccttccc  57061 ttcccttccc ttcccttctc ttccctcccc tccccttctc ttccctcccc tcccctcccc  57121 tcccctcccc tcccttctcc tttctcttcc ccttcccctt cctcttcccc ttccccttct  57181 ccttccttcc ttccttcctt cctcttccac ctgccttcct tttaattttg ctatgagccc  57241 ttaaagagga ttttagtaat ttgctactta aattaaatat atttgctaga tgttgtgcta  57301 ggcttcagga atacaagttg gattgcagta atgtaaagcc ctttgcattc tagcaagaaa  57361 acagatgggt atgtatgttt gctcagtgct acattaaatg aaatggatgg gagccgggag  57421 gagaaatggt gtgtttggcc tgagaggtta gtagcaagga cttctctgca agaaagtttg  57481 aagccaattc ttcaagaatg aacacctttt tgctgggtga aaagtagagg aaggcatttg  57541 gggtaataga aatagcataa aaggtaatga ggtttgaaaa attacatgct gtgtttggaa  57601 gaatgtcctg gagcagcagc gttttagaag gtttttaaag acgatggtga cttgatcaga  57661 gctctgtagt gctttgagga tgggttgaag gtgggcgtac ttggagactg gtgggcattt  57721 aattggtgcc ttccaaccac ataaatgaat gtcccctcaa atcccttgga aacactttaa  57781 ttctagaaaa ttcaaaaatt gtccccaaca tctttttcct ctgagttggt accctggatc  57841 tttgggtctt cttttctttc cttttttgat gttttatttt gggtaatgaa agtcacacag  57901 gttttgaagc cagcagattt ggcttcaaat ccaagtctca gttgcttgct agctgtaagg  57961 gacaaattat atatcttttc taaatactca tctataaaat gggagtaata attgctatgg  58021 cataggattt ttttaaaaaa aagattagaa atcatgtgtg tacagaattt agcacagtaa  58081 ctgatggata ttatttctat tacctgttat cttggtcttc tagttgatag ctccttgcta  58141 gcgtctagct cctttccata gctcttcctg agtagggcca gcatgcagtg ccacagcttg  58201 ctaaggcttc tcctggattg ctgagttgtt ctagtttttg tggcacctca catgctaacc  58261 caccctgaac acatgctctg aaaacataac atttagagga aggttgaaga ctgagagaca  58321 aggtatatct ttgaggaaat tcagatgctt gtcttgagga gctcaggaaa gctagacacg  58381 agtaatgact gtcgtttgtg tgtggcatta ataaatttta caatagctat gtccccattt  58441 agttattcta tgtcacaaat aaaggcagga cagtagtatt tactgtgtta aggtactggt  58501 ttcccaggta tcttacagtg agaagacaga agctcagaaa gtgtaagcaa tgtgcatatt  58561 tggtggagtc tggatgtaaa cagagatctt gatgccaagc ctgtggagct ttgtctccat  58621 ataatgttgt ctctttcata ataactgact gtcatgtggc agattattca tgctattctg  58681 acattgatgg cattaatatc atcttatttt cccaatctat tcaaggatca gttttgcctt  58741 attttatttt gtttcattcc aaattggaga tgtagagaaa aatcacatga agtttgattt  58801 gccagtctcc taaaaggaag aaaaatgtag atttttaata tacttaattt tttttcttta  58861 ataggtatgt gagaaagaga gcagtcccaa attggtggaa ctcttactga atagtggatc  58921 tcgtgaacaa gatgtacgaa aagcgttgac gataagcatt gggaaaggtg acagccagat  58981 catcagcttg ctcttaagga ggctggccct ggatgtggcc aacaatagca tttgccttgg  59041 aggattttgt ataggaaaag ttgaaccttc ttggcttggt cctttatttc cagataagac  59101 ttctaattta aggaaacaaa caagtaagta acaaggagaa tattttttac aattcttatt  59161 tttaatagta tttttttaag tcactagtct tttagtggtt attcatgcca gtttgaggga  59221 ccttaagcca aagatattgc aaaggtttgg attttttttt tttttggcta tgaaatactt  59281 caaaatgaca tttaagttct ttatgagata gcaaatagtt atttataaaa atagagcaaa  59341 atagtggaag ctttttgaag gggtactttt taatatatat tttttattat taaagtaaga  59401 tatccctgtt tttaaaggaa atataaaatt ataaaaaaga aaataaaaat aacttatttt  59461 atctcttata agtaattaat atggatattt ttcctaactt tttatatgct tacatgtacc  59521 tatgcattca aatgtatgta aaagcataca cacatattta tttggcattt ttaacttaga  59581 atatacttta tatttcaatt gataatgcat tttctttata ctttcaagct catgtgtatt  59641 ttgtacatat tatgtgtatt gatggtaagt taccatcttc tgacactatt tttatctttt  59701 gagctctctc atttgttcac actaaatgtg tttttagcgt gaaagctccc agctttccct  59761 gtgttaactt agtcccatgc ccatctcctt ccccatggtc atcaaactcc atgaatcaac  59821 accttaagga ccatcttgca agtaacatgt ttgcttctct catttttatg atgcactcac  59881 tagcaaaaca ccagttttgg tcagtctacc agtctacttt ttccctcagt ttcaccaaga  59941 aaactgagtg ctgctagaga aaagtaccca tccatgcaat ttggtgcctt tatacatcaa  60001 ggtttccaac cgctcagtag gctccaaaag ttccaatcag gctgaatttt cctcggtttc  60061 tcaaacactt cgtgtaccct tacttccagt ctttttccag tgttactctc tctctaccta  60121 gctctaaatt ctctcttcac ctggctgtct cttcattctt cctgtctcag tgctatcacc  60181 agtctggaag gttctcttac atgaccctat agcactttat ttctcacata tactaccatt  60241 caccacatta tataatttaa tttttcattt ttataatcta ctttttggta aattgttagt  60301 accatgaagt caatgtcaat tttgttcatg gttgtaacct taccattgat actagtgttt  60361 tgcacatagt agattatcat ttagaattaa gtattcaata ttggcaaaaa ataaaaattg  60421 tgtaatacat tatgttgata agcatgtgtg gaaacatgct tcatatattg atatgaattt  60481 aaattgtcct cttttgagga caatttggca atatctacta atatttttaa tatatgtaca  60541 tacttttttg acctcacaat gtactgttag gaatctatga tacagacatt ctcaatgtgc  60601 acaaaaatta tgtacaaaaa tgcacattaa aacattgttt ataatagcaa aagagtagga  60661 aaaaaaccta agtattcccc aaaaggaact attcaaataa ataatggtac atacatgttg  60721 tggaatgctt tgcaatcatt gaggaaaaaa aaacgtggag caaatttaat gtcctgataa  60781 agattacatt actccgtggc aaaaaaaagg gcacagacag tgtttttact atgctaatgt  60841 tgatgaaaat gcaactggaa tatgatagtt ataaaagttt gaatatgaaa taaaaccctc  60901 cagaaatggg ttccctggtt gtctctgggt ctttggaaat tactgagaca tggttagatc  60961 ccatgtttca ttacttaaac tagtcttatg ccaaaaacct gcttacttta atcttcaata  61021 tccgatggag aggaattgtg ggcccattgg agagggacag agggagattt atcattcact  61081 atattctctt tgttctgtct ggagttttta ccattgacac atcttaccca gttaaaaaaa  61141 catagaattg tcatttgatt aattggaggg tataaccatg atttcactgg cagctggtct  61201 gagtaaagaa cactttgggt catagctttc aaacattttt caggtagtat ttgcctaagt  61261 gacatatttg tgtgtgagct catcctaccg ggcttcggga taatttccca tatcataaca  61321 tattactctg gaaaaaggaa ccatttgggt atatgggtat agtgtaagcc atagtatcag  61381 ttgccttctt ggggtttatc atatgggtcc accacatatt tacagtagga atagatgtag  61441 atacatgagc atacttcact ctgctactat aattattgct attcctactg ttgtcaaagt  61501 cttttagctg attatctaca cttcacaggg gtaatatcaa atgatccccc accatgctct  61561 gagccccagg gtttattttc ctttttacag taggaggcct aaccagcatt gtattagcca  61621 actcatcact ggatattgta ctacatgata cagcagtatc atgtagtaat atttgaagtc  61681 attacagtag taatatttga agaaatcttc ctggctgtat gtagaacaat gaggactcag  61741 ccaacttatt cttcatagta gagctaatac ataatgtaat gaagttgtga gaagaatgtt  61801 aactttgaaa ttccatcagg tttcccaata gtcataatga atcactcagc aaactttata  61861 aaaataacaa gatcctttat ttagcagttt atgtgttcta tgcattgtgc taaacatttt  61921 atatgcatca tttcaattac tctttttcat caccatatac tgtatttatt atcatcttca  61981 ttttccaggt gaggacactg atacccaggg agctcatata actcacaaat ggcatttcta  62041 tgacttgaac acaggcctgt ctggcttcaa agcctaggcc ttttcctgaa taaagttagt  62101 tccatagaga ttcagttttg ctgtctacat gaaagcattg tgtacatggt tatgtttttt  62161 taaaaaatat atgatctgcc acctgttaat tattcaggat cactagtgta aggtgacttt  62221 gaaaggaaaa atagaaatat tctccagaag catagcaata cgtaagaact ttggtcctat  62281 gtatgtttat ttttgcataa ttgttgattt ctaagttgct ggtgtatctc ttattttcag  62341 atatagcatc tacactagca agaatggtga tcagatatca gatgaaaagt gctgtggaag  62401 aaggaacagc ctcaggcagc gatggaaatt tttctgaaga tgtgctgtct aaatttgatg  62461 aatggacctt tattcctgac tcttctatgg acagtgtgtt tgctcaaagt gatgacctgg  62521 atagtgaagg tatttattat aaaaaaaaac cctttatgct ttatatttac acactgacat  62581 tgaacaatag gacccaagac aaaaacctga cctaaatcat ctggaaaaac ttgagtagaa  62641 atgtgtttat tatcgcaaac agttaagttt actaattttg gttaaagtga tgggtcaagg  62701 aagtgtgtct ctgtgcttct aaatgttata ctaattggtt aatggttaat attccaggaa  62761 acaaactctg actagactgg aacgagattc cacgctctgt cattgactag atcctttcgt  62821 ggcttgtgta agccccttaa ccttgttaaa ggtagtaatg tcgactttgc agggttatac  62881 ataataatta gaaaaaatgt atgtaaaatg tctgcaacaa tgcttggcaa acaggaagcg  62941 cttaataaaa aaggttttta tctttactat agcttaaaac aatattaata ttttaatagc  63001 tcacttgaga taacttttta aaaaattaat atggtgaaat atataatgac aatgattagg  63061 gctgatgtat ttagcattag cagtttggta aaaatggagt gaggggcttt cttattaata  63121 tagtatgatt gaaaacactg ggtgatagaa taaggatatt tgagagggca aaaaatgaga  63181 gttgttccaa aatattgtgt ctcaagtcaa accattttta aaaatcaagt gtagtgattt  63241 atatacatat ataatttata taaaataaaa tgcattcact ttaagtatat attttcatgc  63301 gctttgaaaa ttaacatatt catgtggtca ttgtcgctat taagttatgc aatattttca  63361 ttatccaaaa aagtttcttc atgcctcttc actgaaaaca tccccttccc ctggcccccc  63421 tgacccttag caatcatttg cttcctgaca atgtagatta atgttttctc tagttttata  63481 tacataggat catacagtga gtactctttt gtgtctgttt tccaaaatga ttgtactatg  63541 ttctaccccc accagcagta catgagcatt ctggttgctc tacatccttg tcagtacttg  63601 gtattttcaa gttactttta gctgttctag tggaggttta attcatttag atgtaatttt  63661 catttccctg atgactaatt atgtagagga tgttttcatg ttcttattgg ccattcttat  63721 ttttgtgtga agtgttgaag tattttgctt ttaattgggt tgtcttatta tataagagtt  63781 ctttgtatat tctagataga agttgtgaca ggtatatgta ttgcatattt ttttcccagt  63841 catagcttgt cttttcattt tactaattct atttttaaca aaacagaggt tttaaatctt  63901 ggtgaaatgc agttttccag tttttttctt ttatggtttg tgctttttgt atcccactta  63961 agaaaccttt tcttagccta aatttgtgaa tattttctcc catattttct cttagaagtg  64021 ttaaaatctc agctttggca tttaggtcta aaacatttta agttgatttt tgtgtgtggt  64081 gtgtcaatga agagttgaca tttatttctt tctgtatgga tatccagttg ttccaacatt  64141 acttgttgaa aatattatat aattcctcat tgaattaatg gaagctttgt tctctttaat  64201 tgactattta ggtatggttc tattttagca ttatttattc tgtgccactg atttatacct  64261 tattcttatg ccaataccac actgtcttga ttactctagc ttaatagcag ttcttgaaat  64321 cagatagtgt aagtcctctg gtgttctttt aaaaaaaatt gttcttatta ttctaggttc  64381 tttgcatttc catataaatt tttaatcaac taactttatg ctgggatttt tattgtaatt  64441 aagtccatat ttagatttta tatagaattt atttataaat taaatcatat tacctatgat  64501 tttaatgtaa tctataatat ataattaata cataaataat aatttatata gattatatct  64561 ataaattaat ttgagggaac taatatatta ataatgagtc ttttgacata ttaatgtgat  64621 atatagttca attagtcttt taaaatttct aacagtgtgt tgtattggat gttttctgat  64681 actattatag atggtattgt atttgaattc taatttccag tagttcactg ttgatatata  64741 gaaacataat tcatttattt gtacatgaat tttgtatcct gtaaacttac taagctcact  64801 tacgtgttcc agtaccttat tatagattct acaggatttt ctttgttcac aattatacca  64861 tttggtaata aagacagatt tgctttttct tttctaatat ttatgtcttc tttttttttc  64921 ttgtcctatt gcactggcta ggacctccag tactatgttg aatagaattg gtcagactgg  64981 gcatcattcc ctggtttcca aacttagagg aaaaacatac agtctctaat cactaattat  65041 gacattatct gtagtttttc atagatgccc ttcatcaaat tgaagaagct tcttcctagt  65101 catttttgga gagccttttt ttatcattaa taggtgttga aaaatgctct tcagcatctg  65161 ctgaggtatt catatttttc acctttattt tgttaatatg gtcaaataca ctgactgatt  65221 ttctaatgtt aaaccaactt gcattcctgg agtaaatctc acttggttat ggtacattat  65281 cctttttata tagtattaaa ttttgttttc taaattttgt taagaatttt gcatctgtga  65341 gagatattag tctgtagttt tatacttcct tgtaattatg ggagtaatgc tggcctcttg  65401 aaatgaattg ggaagtgttt cccattccac aattttctgg aagaatttgt gtaaaggtat  65461 tttcttctta aatgtctgat agacttcacc agcaaaggcc atctaggttt taaatttttg  65521 tgaggaggag atttagaatt atgaatttaa taactttgat agatgtatga ttattttaat  65581 tttcttttac ttcttaagtc agttttagta atgtgtaact ttcaaggaat atgcccattt  65641 catataagtt gccaaattta tttgtttaat gttacttata gcaattattt aattatcctg  65701 ttagtgattt aattatcctg ttaatctctt caaagaatca gatttttgtt atattgattt  65761 tctctattgt ttgtgttact ttctcacgga cttttgttct tatctttatt gttccctttt  65821 ccttacttat ttttatttta attgactctt tttatagatt cttaatttgg aagcttagaa  65881 cactggtttt tagaaccttc ttattttgta acagaaacat ttatttaagg ctgtatatgt  65941 ccttataaat atcactttca ctgcatccca atattttgat gtgtcctctt tctattcatt  66001 taaaaaaatt taatttccca aatgtccaac aatgatagac tggattaaga aaatgtggca  66061 catgtacacc atggaatact atgcagccat aaaaaatgat gagttcatgt cctttgtagg  66121 gacatggatg aagctggaaa ccatcgttct cagcaaacta ttgcaaggac aaaaaaccaa  66181 acaccgcatg ttctcactca taggtgggaa ttgaacaatg agaacacttg gacacaggaa  66241 ggggaacatc acacaccagg gcctgttgtg gggtgggggg aagggggagg gatagcatta  66301 ggagatacac ctaatgtaaa tgaggagtta atgggtgcag cacaccagca tggcacatgt  66361 atacatatgt aacaaacctg catgttgtgc atgtgtaccc tagaacttaa agtataataa  66421 aatatatata tatatatata aatttaattt cctttgtgac ttctttgatt cacgagttat  66481 ttagaaggct gtcatttatt ttccaaatat ttggagattt tctggataac tttctggtta  66541 ttggtttcag tttgactttt ttgtgctcag ataacctact ctgatgattt ctatcctttt  66601 atatttattg aaacatgctt tatggattat catatggtct gtcttggtga tgtgccatgt  66661 gcaactgaaa agaaagtata ttcagctgtt attgggtcaa atcaataggc caaattgttt  66721 ggtaggattt ctcaagtttt ctgtgacttc actgactttt ctttctattt attgtatttg  66781 atagagaaat attgaagttg taattgagga tttgttggtt gccttttcag ttttatctgt  66841 ttatgctttg tatattttga agctctgttg tttgttgtgc aaacattaag gactatataa  66901 caaaggacta tatataaaaa taatttttta aataaagtaa ccactttata ttaatctaag  66961 tttgcagtta gtatcatttt cttcctctgg aagaattccc ttttacattt ctagcgcaga  67021 tcttccagaa acccatatgc agattactgg agcacttttt ccataactgc cccagaactc  67081 ccagctatct cagtctccat aaatgttgat ctctatctcc tcatctcagg aaagccatga  67141 gactgtttaa atttcttctt atgtatatgg cccttaattt gtctctaagc agaaggccag  67201 agtgatcata gggctcacat aatttgtttc ccttctttcg ggtattacat agtcctatgc  67261 tgtctgttgc ccagtgtctg tatccagtca tttaatatat ttatccagtt ttcttgttgt  67321 ttatgtgagt aggttaagtt cacatttgtt actctatcat gcctgaaaga actctcttca  67381 agtatttttc aagaaaacat tagaaatttt aacataaaat aattataaaa tgcactatgc  67441 tgcttgtaca aataatgtgc taatagacat ttcacacaca tataaagtgg gataattatg  67501 ataatttaat aatctttata attatgtaat tgttactaaa ttataaatta tattttatta  67561 aaattcttaa gcattaaaat aatttaaact atagaattaa ttaaacttta gcaatgaacc  67621 aggcaggaac tgtttatatc aggatttata ctgaatatta taagggtggt atttggatat  67681 acagatttta taatttggtt ttgacataaa atgaattctt tctttttcta agataaagag  67741 taggggaatt taaaatcata attaattttt cacaaatgca ttcacattat ttatctttaa  67801 tatgattttt ttattgatct ttgttttctt gtaaatttat tattttgaaa gtactgcctt  67861 ttccttattt tataaaacaa ttattgccag ccaaatttat tgtgtttatt ttaataccat  67921 tccataaaag aaaccatgaa acatgaaatt caaatagaaa tttattaaaa attgctgact  67981 gttaaataat ttgtgtgatt acaacattta aagcaagatt tgaaaaattt ataagaaaaa  68041 ttcagggagt taatccactc tctttcctat gctgctagac cctattccag cggtctccat  68101 aaaaaaaatt cagagaaaca gaaagcagta acagtgatca gattgcatac aaactttctg  68161 cacacacata tatttgttat aacttatgta accgttgatt gatttggctt tttcctgctg  68221 gactatgagc tcctctgagg aatagattta ttttttttct gcatatcagt atcccgtgct  68281 tacccagtgt ctagtctgta acagccattc tataaatatt tatgggtgaa gaaaaatgtt  68341 gttgaatttt taaagtgaaa aaccaacatg gcttatcatc tctattttaa agattttaca  68401 aagggaatgg actgtgaaat ttccattaat aaaaataggt ctaatcttcc atgattgaac  68461 tatgatagaa ggatcttatg attgagtaag ctttttgtat tcaccttcat gttattttat  68521 cattttcaaa ataggaagtg aaggctcatt tcttgtgaaa aagaaatcta attcaattag  68581 tgtaggagaa ttttaccgag atgccgtatt acagcgttgc tcaccaaatt tgcaaagaca  68641 ttccaattcc ttggtaagtt aaattgtgca attgtgatta tgttgtgttt tgctgctgac  68701 attctcttga taactaaaat ttatgccaaa gctaggaaca attggtaggg atttccctga  68761 tgtatgaaaa ctataatttt gagattttta tatatgtaat agatatgaaa acatattaga  68821 tgtaaattat gctcaattca catttgtagt cttttgagta tgcagggtat gaattttttg  68881 gggcacatat atatatatat atatacttac agtacacttc aagatggttt tcttctttct  68941 tttcagaact ccatgtctga aaagagccca ggctagacct ctacctaatg gtgtgggttg  69001 gtcccatgaa cactttagct agaaatctga tagtgatttc taagaaacca gacagaagtc  69061 tgaaggacac tgaacaagat ggagtagcat aatataattc attgttcatc tatctatcta  69121 tcatctatct atctatctat ctatctatct atctatctat ctatctatca tctatctatc  69181 aatcatctat ctattttggt gtattgaaag tcatttaatt ttttagatac ctttattatt  69241 atttcaacct cttgtctgtt ttggaactat ggaaggacta tggcatattt gcatgaggag  69301 tctgataatt ctagttgagg aaattgggag ccaccttatt ctcaggttca ctttgaaaga  69361 cctgttctaa cctattctcc aattttgatt atagctgagt actaaaaata tgagggttgt  69421 tttgtgttaa ttctagatct taagatgggt gaaatgaatg actgtagttg aatcggttaa  69481 attagctgtc agtctttata tgctctttcg aatttatata taaatttagt tataaaaagt  69541 agtttggtta atgagaaatt atatggatat agctttttca ctcaaccttt ctgtttttca  69601 gtttccttat atttaaaaca aaggagaaag agtagatgct ttctaaggtc atttgagcac  69661 tgaactggag ttttctttta tcctcataat tgggttctta gtttttactt gcctattttt  69721 tcccataatt ataaatacca ttaaccctat taaaatttca tggttccttc cttataaaaa  69781 tgtcctcttc tccaataaat gacagcaatt ttattataaa ttatttttta ataggggccc  69841 atttttgatc atgaagattt actgaagcga aaaagaaaaa tattatcttc agatgattca  69901 ctcagtaagt atttggatgt aatcataagt aaatagatat tttgggcaga atgcagtgtt  69961 tggttgaatt tcctccaatt attcaaatat tcttggtgcc agtttcatct tacataatct  70021 tcatatatat ttacctaatg attccttcca taagctatag aaaaatgaaa catacattta  70081 aaaatttacc tttcttgaat attatagaac acattagtct ttttttttac aagttttctg  70141 aaatgtaaat aacacctcca ccaaggccac tcttcatcct ctcctccagt tttctttctt  70201 tctttttttt ttcacaccac ttacaccacc tgattaaatg ctttttattt actgttcatc  70261 tctgccactg gaatgtaaac tccatagttt ggttttctac tgaatcttca gtgccttgaa  70321 gaaagcctga ttgctaggag gtgctcacta aacttttact acatgaattt aaattatttg  70381 cttatatttc cattgtttgt ttgtttttga caaaagggtc atcaaaactt caatcccata  70441 tgaggcattc agacagcatt tcttctctgg cttctgagag agaatatatt acatcactag  70501 acctttcagc aaatgaacta agagatattg atgccctaag ccagaaatgc tgtataagtg  70561 ttcatttgga gcatcttgaa aagctggagc ttcaccagaa tgcactcacg agctttccac  70621 aacagctatg tgaagtaaat ttaatttatc cttgtaactt tcaagacatt tgaagagctt  70681 ttgtatttat atctaatttg cataattaag tcgtttaaaa gaacattcta cttttgtgtc  70741 actgggtgat aagtcccccg tgcctctggt ttttgcacac atatcttagt ctgtgtgatg  70801 ttcaggagca tctttgaggg caggcaatgg aaacagatct gattagaaag gaattccagg  70861 ttctgtacgg agtacatgtt aaagtctgtc aagtgtatat tgattatact ttaatcattt  70921 aattcaagta agacaacttc aacaatttaa attagattag gtaaactaga attagacctg  70981 gtttggtagt actggctctg actcagctac caactgtgtg acaatatgaa catgtcactc  71041 cgcctgctct aatccttcat tttctcatct gtgaaataga gattcaacta aatgattact  71101 gaaagttttt ttttcagttt aaaataatgt gtaacttaaa gatttttttc tttttggtca  71161 aagttcctgt cttgtaagaa ttaaagtata acatagtttg ttgataggat agctctctga  71221 aaattgactt tgctcaccat ttgtatgtac tacagatcaa aatagttttg aaagccaaag  71281 aagatatcat aaaagttaaa attattttaa tgcaaatgtt taaattgtta aattctcaag  71341 gctgggcatg gtgactcatg cctataatcc cagcactttg ggaggctgag gcgggtggat  71401 ctttttgagt tcaggagttc aagaccagct taagcatcag caaaatcctg tctctaccaa  71461 aaatatgaaa aattagccag gtgtggtggg gtgcacctgt ggtcccagct acttgggatg  71521 ctgaggtggg aggattgcct gatcctggga ggcagaggtt gcagtgagcc aagatcgcac  71581 cactgtactc tagcctgggc aacagagtga gaccctgtct caaaaaaaaa aaagtcttgt  71641 tgcaaatgca tttccccctt tttaagccta aaaaattaat cataattttg agatgtttta  71701 aaggcaacat tacataaatt ttaagtatat ttaagggatg ttttttctct aaagttttta  71761 tatctggaga cagagagaag aaagaaaggt gacccactcc tccagccatg ccctaatgtc  71821 taaaatgtgt ctttctctct tctccacttc tttgccttgc taaatacttc aagccaccca  71881 gggctcaatt taactgtcac ttcatcactc tctgcttctc ttcctttttc cttctaccct  71941 ccagccaacc tacccaccta gcctccatgt ccataacacc tgatgctttc cacctaattc  72001 tcattcggtc atccaatttg atttaaaata cctttgcccc cactagactg tgaactcttt  72061 gaggacagga cttgtttcag gcttgtttct gtctattccc agtgcctagt gagatctgac  72121 atatggtaga agtttagtac ttactgaatt gatttgtgga ggaataaatg tctgaaactt  72181 ggtaatcctt caattaatat ttgttaaatg agcaagcaaa ataattttgg gatttagtct  72241 agttaaaaca aagagaattg gaagagactg tgacaaggtg agacatgccg gcattcaatg  72301 actggacaag ctcagaacct tctcttaggg aaatttcaaa atgacaccat tagatggcac  72361 tttgtttgtt tgtttgttat tgtcaaaggg tctcatctat gttcctttta taggaacatt  72421 tcctgattaa accttgggaa taattttaaa atctttactt cagaataagt taatgagggt  72481 ctgaaacaaa agcaggaatt ttgaaacaac ttctggggct aagagtggtt aataagcctc  72541 tataatgata tcaaactcta gagtttctcg tgtggataaa tatattgata aataaagaag  72601 accatagaga agtgattgat tttggtattt tagctctttg agagtattac gtacctaagt  72661 tttaaaaaat tgacataatg tgtaagtagg ggtttgctat tatcattata aaattagaaa  72721 ttgcttaaaa atagaaagta gaaatttgaa acaaaaagtt tcgtaaaaaa caggaggttc  72781 taaaatgaaa cacattataa gtaactattt ttatagttaa atcttaaata tatcaaaata  72841 tgtaaaattt ctgacagcat ttaaaacata ttcccaggat tatattgtac tttttgttaa  72901 atcattaatt caaatatttg ttgaggcgta ttctgcttcc attttgctct ttctggaaat  72961 aatttacaaa aaagctgaag gaagctttca actctatttt tgtgaacctg ctttttacaa  73021 tctacctgtt gtaattttcc tggttttacc catgctacaa gcagagacga ttgggccaat  73081 tagtatactg aaattctgtt gtggattgtg ttttcaactt tttgaaaatt cttgatggtt  73141 ctagttacca gaggtgtgta aggcagaaat attagctaga cttaagttcc tcagatggtt  73201 cactttagaa ttttaaacta ttgtcttttc agactctgaa gagtttgaca catttggact  73261 tgcacagtaa taaatttaca tcatttcctt cttatttgtt gaaaatgagt tgtattgcta  73321 atcttgatgt ctctcgaaat gacattggac cctcagtggt tttagatcct acagtgaaat  73381 gtccaactct gaaacagttt aacctgtcat ataaccagct gtcttttgta cctgagaacc  73441 tcactgatgt ggtagagaaa ctggagcagc tcattttaga agggtaagaa agagctcatt  73501 aaaaataaaa gggttgccta aatatgctga tgttaacaaa atatgctgac atttttatag  73561 caatgagttt taacaacatg gtgaaactcc atctctacta aaaatacaaa aattagccca  73621 gcgtggtggt gcgcacctta taatcccagc tactcagagg ctgaggcatg agaatcgctt  73681 gaacccagga ggcggaggtc gcagtgagcc gagatcgtgc cattgcactc cagcctgggt  73741 gacagaggcg agactctgtc tcaaagaaat atatatatat atatatataa tatatgtatt  73801 ataatatata atacatatat tatatatatt tattatatat aatacctata tattatatat  73861 atactatata taatacttat tatatatata ctatatataa tacttattat atatataata  73921 aaagaccgag gcaatgaata ttacaacttt tatcaactga cttacatttt tacaactaat  73981 ttttaaatta atgagtcctc tttgatgctg ttctttgaaa gcaaattgtt tttgatattt  74041 tttctttaaa gcatatgaat ttatgcaatt taatcattat cttgtctctt gtgactagaa  74101 ataaaatatc agggatatgc tcccccttga gactgaagga actgaagatt ttaaacctta  74161 gtaagaacca catttcatcc ctatcagaga actttcttga ggcttgtcct aaagtggaga  74221 gtttcagtgc cagaatgaat tttcttggta agtgttctgt gtgggtctcc tccttaccag  74281 gccctctaag ttgtacaaga tgagtcatat atggaccctt tagttgtgga tttaaaagtg  74341 gcatttcagt ttaaatatta tgctggattt aaaaaataaa attagcaggt tggcaataaa  74401 acaaaatgct ataaaactat gaaaagacat gaaagaaaca taaatgcata ttggtaagtg  74461 aaagaagcca atctgaaaag gctacatact attcgttccc aactataaga cattctggaa  74521 gagacaaaat tatgaggaca ttaaaaagat caatggttgt caggggttag gggagggaga  74581 gatgaataga taaaggacag aggattttta gggcagtgaa actgttttgt atactataga  74641 gggggatatg tgccatggta cacttgtcaa aacccataga atgtgcagta taaagaatga  74701 accctgatgt aaactatgga ctttgagtga taatggtgtg tcaaatattg gctcattgat  74761 tggaacaact gtgccaaact aatgcaaatt gttaataata ggggaaaatg tgtgtggggt  74821 ggtggtggtg gtgagggaag aagggattta ttgaaagtct ctggactgtg tgctcaattt  74881 tcctgtgtat ctaaagctgc cctaaaaata gtctataatt taaaaaaatt atcacatttt  74941 tattgtcaga ggttaaaatg atagttactt ggcctactgt gtagtaccct gtggttccct  75001 ttagtcttaa actaaacatg cacatggctg cctgagctgg gtaaggcatc ctgatactga  75061 gatattgttt ttcatactga agtttcttca gcaacttttt gtatgataaa tatgattact  75121 ctttgctgtt gttagaaata aaattaatca tttaatggtt ttcaaattag tgaagttaat  75181 gtatattcat tcagctcttg tgctttgcaa gacattataa taatgcaaat tattatcatc  75241 acttttatta aaagttgtag aatcccctgc cttctcttag catatgaaat aatagaggaa  75301 attatgttca tttgtatcct aaatgaacat tttaatttta aggaacaaaa tacttttatg  75361 acaataaaca ggaattcccc atattttatc ttccttcata gagaatactc acatcttgcc  75421 acccatgtgt ttattctata cctgactcag taaaataatt tttaattcta tttaatagca  75481 gaatatggct taactactta ttaatagtat tttaatttgt cagagctttc agaaactaat  75541 aatgatcaaa gccatcttaa tttggatagt attttcctcc ttttccgcgc cctcccttcc  75601 tcctaccctc ctgaatattg gccactttcc aaccatgatc ttgaaactgg ggatagagtt  75661 gttgactcac tcactagtaa gcaaagcaaa ctcggtctct gtccttaagg aacttgtgat  75721 taagttcaga gcacagacaa acattaagta atgatggcat aaataaatgt aattttatat  75781 tgtggtgcct tctgtggaaa agtttcagga tgctctgtgg tacctcaggg gaatctgatt  75841 cagactgtgg agtcaggaaa gcatttctgt gcctgtgaca tttaagttgg gtctggaaag  75901 aggagcatcc aaaaggacct tgaagcttgt gtatggtcag tgtgtcatct cagctgaatc  75961 tcacaatgat cctaatggtc atacctagga caggtatgac ctctgcccaa ttgaggaaat  76021 tgaagccgtg agattttttg tgtgatgctt acaggtgcat acaattaatt atcttgttgt  76081 gaaagtttga atggacccca gttcaaaatc tgtttgcttt gcactacaca atcttaatag  76141 tttgagaagt ggtcttacat ttgagaagat gccccatgga ggtgacatct gagcaggacc  76201 actaaccatt aaaaaataat cccatgtaaa tggtgaaacc ctgtctctac taaaaataca  76261 aaaattagct gggtgtggtg gcgtgtgact ataatcccag ctactcggga ggctgaggca  76321 gaagaatcgc ttgaaccagg gagtcagagg ttgcagtgag tggagatcag gccacagcac  76381 tccagcctgg cgacagagca agactccgtc aatcaatcaa tcaaacaatc aataaatccc  76441 atgtaaaata aagttttagt tctgtggcct tatgagtgtt ttccatacag catatgaaac  76501 tcaagactct gaagtcttaa gtggagaatc atttcgattc atttattttg cgaataggtg  76561 aggtataata gctatctttc tgcttctcag gaagacagct tctaggagtg tcctggaaca  76621 ttttgaccct tgaagattgt ctagataaag aataacccat atttttaacc ttgaaaatgc  76681 taataactaa ttcctcaatc cgttttccta gaaacggtag atttgtagat ctctcaagtg  76741 tcttagtgtt catctgatct agctacactc ttttacagat aagaaaagag actgtgtgat  76801 tttctgtttc ccaaattgtg tttcacagaa ttttatttct tggaggtacc tcatactgca  76861 tttccttctt aacaattcat aaagtaggcc aggcacggtg gctcatgcct gtaatcccag  76921 cactttggga ggccggggcg ggcagatcac aaggtcagga gatggagacc atcctggcta  76981 acatggtgaa accccatctc tactaaaaat acagaaaatt agctggacat gatggcacct  77041 gcctgtaatc ccagctactc aggaggctga ggcaggagaa tctcttgaac ctgggaggcg  77101 gagattgcat tgagctgaga tcacgctact gcactccagc ctgggcaaca gagcgagact  77161 ctgtctcaaa taacaataat aataataaaa taataaaaaa taaaaaaatc ataaaatata  77221 ccagagtatt gagaactcag atattttact ttattttact gaatgtttcc caatctctta  77281 agattatatg atcgaagaat acttttatta aataacaccc attattattc catagaacac  77341 tctttatgag ctagtaccat aaagaatttt tgtccttata atatactgta ttttagtaaa  77401 gtgttcatag ttgttttgct atagttattt aagagtaatt ctagttaatc tatatggtat  77461 ataataacat cacacataat ttgaaatgaa tattctagga atttttactt tgtacaaatg  77521 ttgggtagac aaatttatat ttaaatagct gtttagcttc caggttaaaa ggttctggta  77581 agtatagcat attattttat tgtttagtgg aagtattaag gctattacct tatttttaaa  77641 agtggatttt taaaaattgt cagtgtgaga atggaaatca aattaagtga cacactattg  77701 gtagctgttc ttatttttga atttaatgga aatctggtta aaatgattaa aatgtttatc  77761 tcattttttt tcttttagct gctatgcctt tcttgcctcc ttctatgaca atcctaaaat  77821 tatctcagaa caaattttcc tgtattccag aagcaatttt aaatcttcca cagtaagttt  77881 attgttattt taattttaaa agcacattag ctggaacaga acctttagaa acatgatttc  77941 gatttagtca tatagaggta attgatttct aaacctactc aacttgatgt ttttgtatgt  78001 atgaatgatt ttcactagat aaaagaccca actcattact taaaatggaa acttttatat  78061 ttatttagtg gatcattgtg taaaaacaac ttaagattgt ttaattaatt gctgtattag  78121 tataatgaaa tgagattata ctggctatca ctttaacttt taaaatttta attgtttgtg  78181 gaacttgata tgttgccaaa atacccttaa ctttcacatt atgcttaagt tatgtttgag  78241 tgaaattttg ggggaagatt aggcaagttt atgtagttcc aggtttttga gatattttgg  78301 ttaattcatg aaaccagaag cttcctgtta actttaaatt caggcattaa ttggatcttg  78361 agtgtgttgt aatcttaaat gctattctaa ttatatcata catgataaac caaattcata  78421 aaaatatatg tgtaaattta tcttttcctt tgttttcttg ctgtcagcta ttccttcaaa  78481 cactatggct ttttagaatt gacactaaaa tgctgcttgc atgatgctgc aatgagctct  78541 tctgtgagct tatatttagg caaataataa ttagaattta gccatagaga gtgttacaca  78601 aacctataat agctaaatta cgtctagctt tagaatgtgt ttaactgttc taactactct  78661 acagcggttc atctctttaa tcttcctaat aatgccctac gatagccact gttattatct  78721 ccacttcata agagatgaag taacttgccc agagtcatag ttcttaaaca ctggtggttc  78781 aacttcaggc tctcaaatca catgcatact catatgtcaa taatgctagt tttgacgtta  78841 cactttattc tcaccctggg gaaaattatt tgtgatgtta tttcatgtat tttggaaata  78901 ctcatttggt ttatgtcttt tctgtgtatt gttttagctt gcggtcttta gatatgagca  78961 gcaatgatat tcagtaccta ccaggtcccg cacactggaa atctttgaac ttaagggaac  79021 tcttatttag ccataatcag atcagcatct tggacttgag tgaaaaagca tatttatggt  79081 ctagagtaga gaaactgcat ctttctcaca ataaactgaa agaggtaaga cgattattgc  79141 cacttaaaaa atatacttta tgatttgcat cattacaaat tatcatttta agtgatattt  79201 agcttctaaa taccaatttc atgaaactag aagcttcctg ttaactataa attcctgtca  79261 actataaatc cagatttcca ttaaatttaa aaataagaac agctactaat gatgtgtcac  79321 ttaatttaat ttccattctc acaccgacaa tttaaaaaaa tctactttta aaaataaggt  79381 agtagcctta atacttccac taaacaataa aacaatatgc tatacttatc agaacctttt  79441 aatctgaaag ctaaacagct agatataaat ttgtctaccc aacatttgta caaagtaaaa  79501 attattagaa tattcatttc aaattatgtg tgatgttatc atataacata ttatagatta  79561 actcaaatta ttcttaactg ttacttaact atgacaaaac aacttagaac gctttgctaa  79621 tacacaagat agtataagga taaaaattct ttatagtact agctcataaa gagtgttcta  79681 tggaatagta gtggatgtca tttaataact ataaattcaa aataagcatt gtaaatatca  79741 ataccattca attttttttt gttttttaaa caagttgtaa gcctacccta tggtaaatgg  79801 atatggtaac acagcataat ttcctcaaaa aattactttt gtgatatact tttaaaggat  79861 tatatgaata tatacataat tatagatgaa tgtgatgctg tgtgtcattg tatcaccaaa  79921 tctctgtcca atctgttaac agactcttaa ataaaccatt tttctcaagt tgttactggc  79981 ctgtatactg tattacttgt ttttcagctt tccttggtac attcttaaat ttctgcattc  80041 cgccaccatg ctatcaccct aatggatcaa ccttttttgt tttgctccat tctctctgtt  80101 gtaaatctga aattgataat ttgtttgtct cagaaaatat tatttttcaa gttctagcgt  80161 attgcctaca aaaaccaaaa gaattaagtg tctgacactg tgaggttcag caaaactgtg  80221 catatatttt gctacctgat tttttgccag caaatgagtg ttttctatta taaatatagt  80281 atatattgct taaaaatttg gaacagaaaa gaaattactc caattaggat gccacctaaa  80341 gtataagcat gtagctgtac tttgagaaca ctaaattgca tgcaggtttg tagtgactag  80401 gtcttcttgc ctttactgaa ggagcagaat gaagtcacag ataatggata accaaatcca  80461 ttttgtggta agaacttcct tactttcaat gtcttgaaga gatgaagtat gttaccaaag  80521 gagattgggt tttttaatat tacagatgag tgacatagat tgtttgggag taagttttta  80581 tatgtaagtt tttatgtttt taaacacata ctgacaactt atgacaaacc tttggaaagt  80641 tttaaaactc tgttgaaagg ttgtgcaagc tgctgatgga atctgtgagc ctttcttgtt  80701 tcttatcacg ttttttggca gagcacattt cttccttccc accaacaggt tttgcccttt  80761 tttttcccat taagattcct cctgagattg gctgtcttga aaatctgaca tctctggatg  80821 tcagttacaa cttggaacta agatcctttc ccaatgaaat ggggaaatta agcaaaatat  80881 gggatcttcc tttggatgaa ctgcatctta actttgattt taaacatata ggatgtaaag  80941 ccaaagacat cataaggtta gataattttt ttctatttgg ttttactaaa tttatttcag  81001 attttctact ctctgtgact ttgatggaca tatattgtta ctatttaggg aaaaataaat  81061 agtaatattt ggcattaata tgctgtgtgt catttgcctt tcatttaatg aatgtgtttc  81121 tgtggtgcca ctgtagagat ttctcattct tcttagccag actaatgttg agagcggctt  81181 ctcctccttc tgtttctttt cagtggagta gactctaaaa gaaaataagt attgctattt  81241 ggtctctggt taccaattac acaatctaaa gaaatacagc acagtataat aacttctcac  81301 actgtatttc atatagcaac tagttaacat atgcctctta catcttaaag cattatagct  81361 actgacatca tgtgaaatta ctaacttcta ttttgcccat taggatgagt aatctactca  81421 ccttgatcag ttttgaaagc accaaaactt ctcaagtatc actgtttctg gtctttacac  81481 tttaagcact ttaaatatct ttggtaatgg attttatcct cctttttgtt ccctttcagc  81541 acatcggtct tattactttc tcataaaatc ctttgctccc ttttccacag ttactgtatt  81601 aacgttgcag acctcagctc tgtcatcacc tctcaacttg actgtaatat ccaccaaggc  81661 agagaccatg gctgtgttca ctcactattc aaatcttggc acccaacaca gtgcctggca  81721 tacaattaat agttgtttaa ttaccagtga tttatactta ctcattctct tctgcctaaa  81781 atctcttaaa tttatattta acttcatctg tttttatgag gaaggatttt gttttctgaa  81841 ctcctgagct tgatttcatt ttaaaggagt ttgttatctt ttgtgctaat tgtggctacc  81901 cttcatccta cccaattatt tttctctctt gaaactggaa aagatggtca tataaaaatt  81961 ggttcagttc ttactaaaca tttagtagaa ctagctttca gtgtattata ctgtattatc  82021 taactaaata tttttaatat ttaatattta atttaatata taactaaata tttttaaaca  82081 tgtttaacat tttcagaaaa gacagaaaga cctagagcag attagaaatt gtaggcatca  82141 tttgcttttt gaagaaagac attttttcaa atagtggtgc attcttaaga aataaatcaa  82201 gaaaggtaat gttgcttttt ggtcatatca tcaggaatgt tggtcagatt cttattagtt  82261 acaggaatga attgatcact actctgatgt aaaattcact tatgatttag tctttttctc  82321 taatttgaaa ctgtggcaac attttaacat atttcaaaat atatctttct ctatccatta  82381 tatttttgat aacactttga ctctactatt agtttaaagg tggtttttta gctacctaaa  82441 cacttctatt tcattcaggt tttacattaa gatcattagg aatgaaagct aacatctgct  82501 gatagtataa tagtttatat ttatttatga tgttatgtga tctcactatc catatatact  82561 attatatgca tatgtgatat acatgaatat atagctatac atcatatata ccatatatga  82621 atatatacac acacatatat aatgtaacta atatgaccct attatcaagc tttaacagta  82681 tacatatatc tctaccttgt ttctatgtca tatggacttt gtgaaatttt gaactttata  82741 atttataggg tttttctttt cttttctttt cttttttttt ttttttttga gactgagttt  82801 cactcttgtc acccaggctg gagtgcagtg gcctgatctt ggctcactgc aacctctgcc  82861 tcttggcttc aggcgattct cctgcctcaa cctcccaagt agctggaatt acaggcacct  82921 gccactgttc ccggctactt tttggatttt taatagagac ggggtttcac tatattggcc  82981 aggctggtct caaactcctg acctcatgat ccgcccacct cggcctccca aaatgcaggg  83041 attacaggtg cgagccaccg cacctggcgt ataatttgta gggtttttca tactatttaa  83101 agacattaga atatgtatac atgtatgtat atgtgtgtat atatagaggt atatatatat  83161 tgcatatcgt attctaatta gtattgcaaa catattttgg ccttttgatt atttctggtg  83221 atagtgtaac atgttttctt tggtgatttt accaaacatt atcaactacc ctaaaatctc  83281 tagcaaaata tatgcattaa cagtactctg aaagacatgt acattattag ttatatgaga  83341 tatgcactct tctggatact atattttaga atagtgtgac atgtaaaaga actcacctaa  83401 atctcaagta tacttttaag cagtttatta ttttattttt atctttcaaa tactaggttt  83461 cttcaacagc gattaaaaaa ggctgtgcct tataaccgaa tgaaacttat gattgtggga  83521 aatactggga gtggtaaaac caccttattg cagcaattaa tgaaaaccaa gaaatcagat  83581 cttggaatgc aaagtgccac agttggcata gatgtgaaag actggcctat ccaaataaga  83641 gacaaaagaa agagagatct cgtcctaaat gtgtgggatt ttgcaggtat ttctttctat  83701 agaattttaa aattcacttt taccatttgt ttggaacagg gattcaaaaa ctgagctttc  83761 tgttctaata tccagaaacc tggtagactg tatggaatta ttccaaagcc cttcatttct  83821 cctaatttta cccttgcctc cagaatggag aagaacatgg agggatatgt taggaacaat  83881 ttggtgctag gtactttgat cggttgctga caaatatgct aaaagtggtc aatcctagta  83941 aaaacccaga atagttctct aaacatggtc tgttgttttt ctcttattag tatgctaaat  84001 aataaatagt attattctcc cagatttttt tttaaaaaag gattcttgcc tgtcgtttga  84061 aagattaaaa aaatttgtct ctaatcttta tttaggtcgt gaggaattct atagtactca  84121 tccccatttt atgacgcagc gagcattgta ccttgctgtc tatgacctca gcaagggaca  84181 ggctgaagtt gatgccatga agccttggct cttcaatata aaggtgattt gttctgatca  84241 tttgaaaata gaaaataatt catgtgtctg tgtgcgtgtg tgtgtgtgtg tgtaagttaa  84301 tttattttgg gcaaacaatt gcttcagtct ctttaaatac tttcttaaaa gaagcactaa  84361 aattttgaat tgggaaactt tccgagtaat gaagtcataa catgaaaatt gtatgttcca  84421 tgttggtgaa tgttattggt aacctgaaac tcttttatgc tgtaaaactt gaaaatatat  84481 atgttcaact gttttttaat tatattattt cttaaatgaa atctaaattt ttctaattta  84541 aaataagcta tattaaagaa aagcaatcta tatatatata tctcatcaac tttgtactca  84601 ggggccattt agtgtgaaat tcttcagatt gtatccttta agtggtccca gattattatg  84661 ctgttacatc tggaatctcc cttttgttgc ttttctatct tttcctttgt tgtcttgttg  84721 tcagctattc cttcaaacac tatggctttt tagaatggag actaaactgc tgcttgcatg  84781 atgctgcaat gaactcttct gtgcataaag tccttaaaaa gcttgtgtca ggacatttaa  84841 ccatgtaatt ggctgcatac atgcttgttt tgtaatttgg gtatttttta atgtttcttt  84901 tattaacttt tttacagcta gccaacgtga gcaaatagta cagtggcagt catatttgct  84961 tgagtggctt ttattctttc attgtagact ccaaattggt tgactttaaa acgaatttag  85021 aagattaaat tcacagataa ggaagagaaa atataaacta tatgacgtta atttgatata  85081 atttgtgggt ttatgaaatg cttattttat ttaggagtga ataactcatc ttaaggcatg  85141 aagatgggaa aggaaaacta taccactacc gttatatatg ccacctaaaa gggtgaagaa  85201 ttgggttaag aaaggccaaa aatgactttt taaaatgtcg taaggttaca tttttttctt  85261 aggtttaagg aaaaaaggac agttgttctt ttcttcttct gaagtctgct agtttctctt  85321 ttccattcaa gtgaatgtca cggaaagcaa atatcaacag gaatgtgagc aggcccagtt  85381 tgaaagcaaa cacaagaggg ttttgtgtct ttccctccag gctcgcgctt cttcttcccc  85441 tgtgattctc gttggcacac atttggatgt ttctgatgag aagcaacgca aagcctgcat  85501 gagtaaaatc accaaggaac tcctgaataa gcgagggttc cctgccatac gagattacca  85561 ctttgtgaat gccaccgagg aatctgatgc tttggcaaaa cttcggaaaa ccatcataaa  85621 cgagagcctt aatttcaagg taacatggta ggctggtaga gaaatgtaat ttattgattc  85681 tcaactgcct agaaatgtca gaaattttga gaagtgagca actcacttaa aattgtgggt  85741 tttctttcct tgttgctgtt agcattatta aagtcctttc cattttaaaa ttatttatgc  85801 cagacttcat ttctaattca tagaaatggg aacaaaaaat aattagagga acctgagaga  85861 aactaagaga ccgtttctgg gatactgaga aaatgtttct gagagagaat ctgagaaaat  85921 gtttttgatg ccttttctga ttcaacttct tatagtggtg attcaatcac aagggtaaag  85981 gtgaatactg aggtcttggg atcatctttc ttctattatt ctttaactgt tatttttcca  86041 tttcctcttt tcttttggaa ttcctgtttt atggacatct tgatcttttg tgccactcat  86101 tcatgaattt tgtcactgtg attcccattc caattttttt ccctccgtat tgtgaggcag  86161 ctgttttatt tagtcatgaa gaccactaac ttggttttca gcagtgtctc actaattact  86221 tagttcatac aaaatgggct ttttatttta ggaattatgt tttaaatgtt taaagttatc  86281 ttctcgtaag ccaaattttt ataaaatgta aataaatcag ttatcagaga gaacactttt  86341 ttttttaaat acttggcaga aaaaagaaat cttcactggg tactacaggg agtgtggtgt  86401 aaactgtact gaaaaatacc cttgatagtt ccatatgaca aacataatga tgaatttcac  86461 ttagtctgtc ttggcttagc tcaatagcac taatgatcaa gatactggct gataaataga  86521 gtcctatttg gcctgggcag tcccagcata attatgtaat agtgtcccac tatattctca  86581 aaagcattcc aatttggatg ataaattata tagtcacctt ggttataact ccatgctggc  86641 cagttagctt agttctgttc catttatata gattatgtgt gcttcactcc aaaacctaat  86701 gagccatttg taaaagtgat ggcttttgcg gtgcccaggg agagaatttg tatgtttgta  86761 tccttcaaca cacatttatt acagttatta aaaggtttta ttgatgatag atggtaatgt  86821 catgtaaaaa tgacatatta tttatttgta gactttccta ttctcttgtt ggacatgtaa  86881 ttagaaacta atatgactta aagaaaaaca aatacacaaa atttattcat ccaattaatc  86941 tcttaatcca ggtgtttttt ttttctgaga ctatacccat acttcaataa ctttgttgtt  87001 actgagaata ttttgagttt ccctttttgt cattgttgtc agagaatgta tcatatcttt  87061 aaaaagactt gttggaggat gagtttgttt tgaaaaggcc tgaatttagt tgatgcaaag  87121 tcacagataa gatggttcat taagctgtat taatactgct tttgtctaat agatatcatt  87181 accaataagt cagactagtt tttcttttgg cacttataaa tcacctttga agacaacttt  87241 ttacaaggaa ataaaacaaa tgctttgaga aataccagta ttattgaaag aaaagtatat  87301 attgctaatg gatgcagcat tctggcataa tggtttgaaa actcatttga ttgctttgta  87361 gaagaatgac tctttcagat gacccagggc ctgtgagcct gccagaactt gaaaattctt  87421 tcttccctga ggtgcttcaa cctgaattca aagagcagct tttaatctat tagagatcat  87481 tttttgtcct ctcatttatt tttcatattt gcctttgatc ttagctcttc tctaatcttt  87541 ttctgtctca accttattaa caggtgtctg tgcagacact tttaagtttt gttttttggc  87601 tcagcctgtc agttaactga taatcatgct gaaaggagaa gcaggacaaa acagagttca  87661 atgctgacaa tactcctttt aatcttgtcc agcccattag cagagcaggc atctctgtgg  87721 gccttgagac gtagtcccgt aaaactcatc ccgtttctac ttgatttgct ttctttgaga  87781 actcttgttt atttttatat ggaggtttcc tgccttggat taaaacataa acctcaatct  87841 gaagttcaat ttcatcttaa tttatgaacg actaagagag ggaacatgaa aagtggaggt  87901 tagtgaaatt atctctaatt ctctgggtta agagatacat gaaaacagtc tcttgagtaa  87961 ccatttgcag gtaaatatgg aagtaatggt tatggttgtc tctttaagtt tttagtcaca  88021 agtagaaaaa gaccaagtta atttttttct gtgtgtgctg aatttctatt tgtagtaagt  88081 gtaagaattt aagcagaaat tctgattcgt attttcagat aaaaagaata tgtaatttcc  88141 ataggtccag aaatagggag agtttgccat ctggtggttc ttaacggcac tctggatatt  88201 attaagagtt gcatttctat ttaaaattat attttaaaaa acgtttggaa gatactttta  88261 ttgtagaaac tatcctctta gggccattct ttaaaaaaat cttattttat atatttctca  88321 ttttgttgat agtgattaga ttctaagagc aacagaacaa tgatcatcct ctcctatcag  88381 aatcactgat gtttagatga tttctcattt tcccaagttc aaggttccat gaaaaacata  88441 gcttgagtgg gattttatgt ctctgcgttt cactgttgat atatatgtcc tcccaatata  88501 acattttaca aataaccaag cacaaaattt aatattttac cttgaatatt taaaatataa  88561 taatatccaa aagctcttgt aatttgtact gatatcttat actagcgtgt ctgtttcaca  88621 ttaagtttaa tgtcttagga tataaaaaat cttttttatg gttagtgatt tatcttgttt  88681 ttttttccat ggaatttctg gatagcgaga taaatatttc catactattt tatttgatat  88741 ttccaaattt gcctctgaat caacaatttt cctattttaa tttcattgta cttgttcctt  88801 acaacctaaa tagcttttta ttatattttg attttattta aaaatgtact tctgaataat  88861 atatctgttt ctgtaaaaac tgttagcact gaatttgcca accatttgac aaatacacaa  88921 ataaaataga tttttacggc ttgtcatttg taatttcata gatccgagat cagcttgttg  88981 ttggacagct gattccagac tgctatgtag aacttgaaaa aatcatttta tcggagcgta  89041 aaaatgtgcc aattgaattt cccgtaattg accggaaacg attattacaa ctagtgagag  89101 aaaatcagct gcagttagat gaaaatgagc ttcctcacgc agttcacttt ctaaatgaat  89161 caggtttgtg tttttcgttc cttattttca aagctcagct gtagtaactt ataaaagtgt  89221 ttctgaatct tttatagaat ttacattcaa agttgagaga atatccatac ggttctttaa  89281 taggccactg atttttttct ttttggaaga tcatcatgtg tgttcatgac aaatcatgta  89341 tcatgtcata agaaaacaaa tttagaaatc acctaggagt aaagcagtgg aaagagtccc  89401 tgagtgggag ttaaaatatt tgggttctag aacttgtctt tactattcag gagctgtgga  89461 accctgaata gtcaaatgac attcataatg tcaaatgagt ttagtgcatg tgaaagttat  89521 ttttatattg caaaggggaa ttattgttgg catggtctaa ctgggacgct tggagagtca  89581 atggctccct gagatgatgc agcttctgag tggaagatct agctctcttg catcaaatat  89641 tgatctcaaa gatgaaaatt ctcaaagcaa cttcagtgct aattgtgtac ttgatcatat  89701 taccttgcta gaaatgtgtg agttgtttga tagtactaga gtaagtgact gggaagctgc  89761 ttttgatccc tagattctgt tgtataaaaa atagcttccc gtggtttatg atctgttcct  89821 tttccccatc gttcttaagg tatgctgaga tatgctgtgt ttcttatctg tatttgaaaa  89881 taaaacatgt ctttgtagtg tgtattcagc aagcgaaaca gaaaattatg aatttctact  89941 tatgtgtgaa atatgctctg taatgcatgt cagtgtctca aatatgctta aatatgatca  90001 ttttatgtag tttaaaaata ctccattata atattggaac tttagaccat aggatgcaca  90061 gcttctagtc ccagctctgt cactagctat gctgaaattt cttcacctgc aaaatgagga  90121 agttggacta gattttttct aaagcccctt gatatttgtt ctagattcca tgtttcactg  90181 tttgatgact ttttactaca ggagtccttc ttcattttca agacccagca ctgcagttaa  90241 gtgacttgta ctttgtggaa cccaagtggc tttgtaaaat catggcacag gttggtgtct  90301 tttatttttg tggcacgggg gttatggtca aagcatagaa cagatggcgc ccagagcatt  90361 gagcatttta gaatttgggt ttagttaagg cagaaacttt tgtgaatttg gaaaactgtg  90421 gaacatttca catagaagac tacttgaaga gcttcatgga agaaggaaag atgtcttgag  90481 ttcacttcca tgacttggtt ttcaagccac atacagatgt ttgtatcact ctgccccatg  90541 ctgctttact agatcctgat gatgtcattg gtttggttac tgaattagtc aattgaatga  90601 tggctttgtg gaaatccttg gggtaaacac atataagaaa attaggttgc tgagcctgtg  90661 aaacctctat ctagataaca tggaggtgag ttttgactta agtgaaatga tctgagcttt  90721 aaatgcttac gattttgaaa actttggatg gccttggtta tagctatttt tttcttatat  90781 ttcacatgga aaatgatttt tttctccaaa tgataatcca ttaccaatga gtttaattag  90841 ttataataat ccatctctgt agctttgaca taaaagacca tttgagcaaa acatactacc  90901 tcagggcttt tcaaccccag catgatgaca ttttgggcca gataattctt tgttgcacat  90961 tgtaggatgt tagcagcatt tttggccttt atattcgaga cgtaagtagt atcctctagc  91021 tgtgacaagc aaaaatgtat ccagacattg ctaaatattg cttggagaat gtgaaaaatt  91081 accctagttg agaaacatta agctactgat ttgttgatga gtaaaattta tagttttgca  91141 tgtggctgcc cgagttccta aaattattat atatttttat gttagaaata tctcttccaa  91201 ttaaaccata aaggtaatta aattcactca ggcagccttg aataattgtt cctaaattcc  91261 atctaaggaa aaaaaggaag ctattgtgaa gagagaactc agttgaggct aaatcctgta  91321 ccatggaact caagagcata ttgaaacatt gcaatcagca attatttgca gtgtgtcagt  91381 tattactatt ttggtaggta tttttaaatt agattttcag ccttctgcac atatgtcatg  91441 gataatgtga ttttactcaa ttattaaatg ataatggaga cagtagtgtg acccagagca  91501 cttacttgag catcagcttg acctacgttt cagtctcttt aattacttat tagctctgtg  91561 aaatttctta atgcattaag cctttgttta cttacttttt aaataaggaa aataacaatt  91621 atcttctata ttgcctccct ggttcagtgt aagtgagggg taaatgttag ctaattttat  91681 attggatcta tttggcaatt taaagaatgt taatcaggaa attttaaaaa attcagaact  91741 ataaagaggt acttacgtag ttttggaaag tgtgtcatgt atggggacaa ataaaaaaga  91801 tgtgtaggta gctgcatcct gtacagcaaa ggaagtttta aatatatcca gcaattttgt  91861 tgtcctagct ggcgcacaat agttatcagg aggtaactca actccacata gtcaaggaaa  91921 agctaaagtt gctctctaaa gtggtgtgtt tccatgtcac tatggaacac ttgaagttgc  91981 acacatgtga acattaggat gggtatatct tatacagtag aataaggaag aggtttgcat  92041 cagaactccc cttttaaaaa aatgcagatt ttcactatga ctgcaataaa attcctgaag  92101 attctgtgga gtaattaagt tgaaactcca tgaaagttct tctcattagc atagttataa  92161 atatgataat ttaagtaaaa attaagttaa tttgagccac tcaaagttac ttttaaagac  92221 agatttaaaa tgtcaataaa atgataattt aaatttccga ttaacctaaa aaagaagtgc  92281 catcattttt atttatgcca ataaattgaa atataatgtc attttatcac taaggtttaa  92341 aggaaatgaa atctctaaat aatcaagtga aaccaagagc aacttgtctg acagctatta  92401 gcaaaaataa ataggagtat tcaccttcat gaatcaaggc aagggccgga ataatttcat  92461 ggtgcagaag ctctaatgag cccacccact ctatgcgccc cgagctgtta ggtcactaaa  92521 cttattaaaa aaaggtacca ttaaggcagg gagaagttta caagactcat ttaactgtat  92581 gataaaagag atatgaaaga gacctattca attaatcagg tggaacatta aaaagcttac  92641 atggcaattt aaccttgata aaaatacatg ggagaaatac aaaggaattt ggaaaattct  92701 ctttccttga ataaggcatc agttagctat tcaggttatg aggttgaagg aatgttagga  92761 gctcttttaa aggtgataaa gtcaagataa tgttgcagat tttattctta tgtaacaaac  92821 cccctcgaaa cttggaggct taaaatgtga acaatttatc atttctcgtt cttctgtggc  92881 ttgactgggc tcagctgcgt ggttctgctc cacatggtat tggcaagggt tattcacttg  92941 gcttcattca ttaaactgag ctggaaagtg caagaaaggt acatgcatgt ttttggagta  93001 ttggtgcttc tccatgtggc ctatcatatg gctaagttgg gcttcctcgt ggcacggtga  93061 tcacagaata attagacatc tttcatggtg gctggttacc aagagaaatg aagcagattt  93121 tttctgtcct cttaaaggct aggccaagga ctggcaaaaa tattaattct gctacattct  93181 agtaaccaga gcaaccacaa acctagctca gattaaaggg gaaggaaaag agactctata  93241 tgaatagcac ctatgtatag ggatggaaat gatgtgtcca tctttggaaa cttccactat  93301 aaatagtggt agcacgctat agatccacta ggaaaatcaa gcacaaactc tttaaaaaat  93361 aagtgtatct tagtaaaata gattagaata actagataat aatggctaac atacatgagg  93421 ttaatatgtg cttttcaaag attagctcat gtaattctca cagcaacctt tccaaatggt  93481 actttattag cccctatgat acagatgaag aaattgattg acagagaggt tgaataattt  93541 atccaacggt acacattcag gaagaggtag agttagaatt tcaaaccaag tagtttgact  93601 ccagggccta tgagtttata cattcatagg gctgatattc aaatgagaga agagaagtaa  93661 taaataaaca tataatatgt tgagtggtac agagtgctac aaagaaaata tgaagtgcag  93721 ttggagatga attgtcaaaa aaggtcttag cacttaaaaa acactaaaac agcaaacaat  93781 tctctttacc acctaaactg taagagcgat ctggaattgc tataaagtac acaacatggg  93841 agaagtctta aacaacagtt tttattattt ataggcccat tgcacactgt cattaaatac  93901 caatatgttc aatcaaccat gcattcattg attcaataaa tactgtacat acaaaataga  93961 aatacagaaa tgggtaagac aagtccttgg gcctaaggac tttataacct ggtatttcac  94021 tcaactacat gatagcataa ataatgtttg cttctgttta agtattcctt aaacattata  94081 gatctcccaa agaaaattaa atacaaacct ctttttaaag tgaatttgac aaagcaaaat  94141 aaattggaat atatagataa atatgctaaa atttgtcata tgtactttgc gtactttaca  94201 tgtgttattt cattctcagg gcaatctaag acagtcactt ttattatctc attttataga  94261 gaagaaagct gtgcagtaaa gaaatcaaat acctttccca aggttacaga gctagtagta  94321 gagcctggat ttgaatctgg gttctgactg atttttaact gccatgacaa ggatcaaagc  94381 tcaaagtgtg atctctgtgt tagaaacatc ggggttgctc tttaaaaaag ccgattctca  94441 ggcctcaacc cagacctact gacccagaca ctgcaagtag aatccatcaa aatgcagtag  94501 ttactttgag aatcatgaaa ctctgctaca cagtctgtct tcctattcat ggaagtcctc  94561 tcctagtata taaatgtgaa gtaatatttc tatttcaaac ctgtattgat aactgtctgg  94621 aagataattt tcctgggaat atattattga tgagactgca aaacagatgt gaggtattgg  94681 attgatcttt ccattgtagc tagggaaata ctgatgttca ttgtttcagt gaagttcaat  94741 gatttcctat ccgaattaac tcccttaatt taacaatttt tttttttttt ttgagagtga  94801 atgcccctct gggcttctag gccacatggt tgctagagaa attaggtact gtgttgcact  94861 tgaaaacact aaaatctttc tgactacttt cactgagcaa agagacataa aatgctttaa  94921 atttgcaaca tttcagaaaa taaattttag tgattattta tgactcgaat ctttcagatt  94981 ttgacagtga aagtggaagg ttgtccaaaa caccctaagg gcattatttc gcgtagagat  95041 gtggaaaaat ttctttcaaa aaaaaggaaa tttccaaaga actacatgtc acagtatttt  95101 aagctcctag aaaaattcca gattgctttg ccaataggag aagaatattt gctggttcca  95161 agcaggtaaa gaaaacctta aaaaattaat tgctacatgg aaattcacta tctattcttt  95221 taattgtcaa actaactgta gtctataata gatgtattaa ataaataaat atattttgct  95281 tctagtgtaa acctcctact gacatgtatc atttattttg gaataaaaca ttgcatctga  95341 cactttaaca atatagtaaa tcacttactt tatgtgtata gttactagtt ggcttatcac  95401 tgttgaaatt atttaagaaa ggtaaatagt ggagattaat gtgtgtgtgt gtctgtgttt  95461 gtgtatgtgt gtgttcttaa acaacactga gagagtttat taagcaagtt ctgagaagat  95521 agtgagtttt caacagaatt ttaaaagcat ttatggcatc acaatggatg cctatgtttt  95581 agcctatact atggaaattt ttcctactgc tctaagcaac tgggaaattt ataaagtaat  95641 atgatgttga aatgtgcaaa ttacattgat tgatggatgc agccaatttt aaaaataaat  95701 atacactttt tttctaggac atgtattttt caggatttat ataagattac atttgtctat  95761 gcataactaa ttgtaataat ttatgtatta gtgcacaggg attaccgaaa atatttcatg  95821 catctacatc tgagcatgca tttgaattgg ttattgacca ctgaattttt ggtgtaggaa  95881 aaatatgtag tgaaacaatg ttacaaaaag attacaattg tttggaatga ttaccttcat  95941 tgactttaag cagtaaaatc atttgctcaa caaggttggg tgttttgtga ggctgtataa  96001 ccatagtgtc cttttgcctt tagtttgtct gaccacaggc ctgtgataga gcttccccat  96061 tgtgagaact ctgaaattat catccgacta tatgaaatgc cttattttcc aatgggattt  96121 tggtcaagat taatcaatcg attacttgag atttcacctt acatgctttc agggagaggt  96181 aagtatctaa tgaagactta ttagattttt agagactatt aatttagact tattaatttt  96241 tagagaaatt agggagatgg catatgaaaa gtaatatgcc attttctcag agtttacttg  96301 tttggaaggc agctgaagaa ttagaaaata agctcataaa accttggagt aggcaatcta  96361 aagacacaca agcacatata acctcatcta atttgtcagg aagaaaattc cttaggtgct  96421 cactcagatc ttgactgtga ttacattgta gggactgtaa ttatctcttt tctgttgcac  96481 agccactaag acatttacaa aaaaagagca aatccggtgt ttataatgct aactctttct  96541 tctaaaataa atagagacat tttggtactc caaagggaaa atatcatttt ggggattaaa  96601 attagcttta cacaggtgtt actggtttcc aaaataaacc ttaccttgat tggaattaat  96661 caacatatag gtagttacat tgcattaaaa agttcagaaa gttttgcgtt tagcatgatc  96721 aaaaacttct ttttaaaaat tatgaggatt tatttatgat tttctttctt catctgtcga  96781 gcatattaaa ctgcttaaca gcatcaacct gaaatggatc ttaatgtgca ggggatttaa  96841 ctctttttat tgtaaagttg tggataaaat atttaataga tatggatgag gactcatatc  96901 agtaacaacc caatacttta tttcaaaatg aatagatctg tattacaatc acttgtgttg  96961 tgtgcagtag attttttccc tttaacttag gaagcagtta ataattaatg gctccatttt  97021 ttagaacgag cacttcgccc aaacagaatg tattggcgac aaggcattta cttaaattgg  97081 tctcctgaag cttattgtct ggtaggatct gaagtcttag acaatcatcc agagagtttc  97141 ttaaaaatta cagttccttc ttgtagaaaa ggtaaggaaa tcaatttgaa tgttttcaat  97201 tgcaacacta aagaaattta aacttaaaaa aaaaaaaaac tttaccttaa agctttgcga  97261 cagtatgagg tttagacaag gtgttgagct ctgttttgaa tcatgtaggc tgtattcttt  97321 tgggccaagt tgtggaccac attgattctc tcatggaaga atggtttcct gggttgctgg  97381 agattgatat ttgtggtgaa ggagaaactc tgttgaagaa atgggcatta tatagtttta  97441 atgatggtga agaacatcaa aaaatcttac ttgatgactt gatgaagaaa gcagaggaag  97501 gtatgttttg atacaactta caaatgcttt taagtgatcc ttcaatactt atgaagtgac  97561 ttttaataaa tgtaaatatt cttatccata agggatgagt tgaaaaatag tatattcaat  97621 tatagggaca gttcagaaaa ctgaattata tttattacca ataaaatctt gtattctaga  97681 ttcagaaaat gttgatttga gggtttgaat gctggcttat tgagcaacat aacctcatct  97741 gtgaaaccgg aataccaacc acatctatct catagaactg ttataaagat tcaaatagac  97801 aatacatgga cctaatttac caacatgtct gccatataat aacagctgca gcttcatgaa  97861 tgtggcaaaa gcagagagta gataactttc tagtcagatg tctggtagtc tgcagcagtt  97921 cagaattcta caagtgaacg taggaataag tttttaaaat tccaagtaga tagatactaa  97981 gtgaatcttt aaaatgttct caaatttcct agagaaatat aggattggtt agaaagggag  98041 ggattagaaa ttatagaaaa tattccatta ttttttcaca tcaaaaccac aaatttatgt  98101 atctccttaa atgttgtttt tatttaaaaa atgttttatt acttctcagg agatctctta  98161 gtaaatccag atcaaccaag gctcaccatt ccaatatctc agattgcccc tgacttgatt  98221 ttggctgacc tgcctagaaa tattatgttg aataatgatg agttggaatt tgaacaagct  98281 ccagagtttc tcctaggtaa ttctttttgt taatttgaga ataaaaatta ggatgtaatt  98341 ttctccttat aatttagaaa atagatttca taattatatt gtcatagatt ttactgtctt  98401 catatatttg ttataatttt tgtatttgga atgatatatt ttaaaggaat ataatattac  98461 agatctggaa tttgttttgc acataatcat gtagactagg atcaagatga ggatgagatt  98521 atcatggaag cagaaatatt tatgaaatat atctttgtat ttgccttaat tgccagggat  98581 atgggaggca aataagacag ttttcaggtg agttaagtga agcagccata ttttataaaa  98641 tgacagaata ggtaaaggaa gcacacctca gtgtagccat agcaggggtt ttatgactca  98701 gtgtgacaat gctgaattct catagaaata ttcattaaaa gccttgaaat taaagtcaaa  98761 agtgttacat ggtgacatac tcaaatactt tttttttttt ttttgatatg ctgaacaatt  98821 tacatttctt ggttccgtga attcaatcag tgattttcag tagagtatga tggaaatcat  98881 tgaattcatg tagcatgttt aggtgctcat tgagaaaagg tgaagtcatg gtaaccatgt  98941 ttcaatattc tcatttgtat cttgacttcc tgcacatgga tttttgggcc taaaagatgt  99001 ttttaaaaca tgctcataca cttcagaaga tgaaaagtgt atgcattata actactttgg  99061 gaaagaaaca gtcaacatat gttactgtat gtcattctgt agattacatg tgtggtttct  99121 catgtctctc agaataaaag ctaatgtctt tacaagacct gcgatgctgt gatctgtctg  99181 gctcctcggt tatcattttt aaaaaaagat atactttgta caaatttttt taattgacaa  99241 gtaaaaattg tatatattta tggtgtacaa catgatgttt tgatatatgt atatgttgtg  99301 gaatggagaa gtttagctat ttaacatata cattatctca aatatttatg tggtgagaac  99361 tattaaaatc tactctcata gcaatttaca agtatacagt atgttattat taactgtagg  99421 ctgacatact caagttttaa acattcctga gagtcattgg gacaactatg aaatgcatta  99481 gattgattta atataaagca tttgaagaca attttgacct tactttgttt agtttttgtt  99541 gttgttgtgt gtatacattt aattttaatc aaattacccc agaaataatg cctaagatct  99601 gtcagtcagg acataatatt attagcaaaa agttgtccaa aatttgagac atgatattta  99661 aagctaaata aactccttta tacccctctt attggcattg attgggaagt ttaggttgaa  99721 tttaaatgct ttggaactca ggaagttaat gtattagtaa tagtgggtta acataaaatg  99781 ctgaattgtc cttgctgaat cctacatctt aaccccagac ttcaaggtat acaggaaagt  99841 accagacatg gtgcatcctt cctctgaaga aatcccaaac tgtcagacac agatccctaa  99901 aatatttctt tttcctgcat taaaatgtgt ttcagatgaa tggacacgtt ttgagtagtg  99961 tatgtggaaa cgtcatttac aaagtctgtt tagttggcca ggtgtagtag ctcactcctg 100021 taatcccagc actttgggag gccgaggtgg gtgtatcacg aggtcaggag ttgaagacca 100081 gcctgaccaa gatggtgaaa cctcatctct actaaaaata caaaaaaatt aactgggtgt 100141 ggtggtgggc atctgtaatc tcagctactc gggaggctga ggcagagaat tgcttgaacc 100201 tgggaggcgg aggttgcagt gagccgaggt tgtgccactg cactccagcc taggcgacag 100261 agcgtctcaa aacaaaacaa aacaaaaaac aaaaaagcaa agtctgttta gctacccata 100321 taggaaaatg tttgtgatta ctctcccttc tctagaccca tgtcccataa atccataaat 100381 cccatgttca tttacagaaa gcagtctaga taggagtttc tcagtctttg agctgttgcc 100441 attttggctt ggataactaa ctctttctta tcgagggtca tcctgtgcac tgcagaatgt 100501 ttggcagcat ctctgtctat ccactagatg tcagtagtat ctccccttcc ctcagatgtg 100561 acaatcaaaa atgtctccgg atgttgccaa agataagggg tggggttgaa taccagtgat 100621 ttaaacaaat taggtgtatc cttctaaaaa cattttacag gtagcgactc cagcatcttt 100681 atattagagt aatctggaga aggttatgcc tctctcaatt ttccctcttt ccatttttat 100741 ttgtagggca gcaatgcatt caggcttttg gtaactcttt ttcccaagat agcagtaact 100801 attatgcagt gagtaatacg acccacctta atagatatga atagacttgt tttgtgaata 100861 tattttaaaa tataaatgta tgggattctg ttcatgcgtc tgagaagcca cagggtacat 100921 ttcctctttg tggagctatt tatttttctg gagagccaag acaggtattt ccacttcagt 100981 ggtgtgattt gaggggttag gaaaatttcc ttgccttcaa ttttctttcc aacctagatg 101041 tcacaaatac ataatagtag tccttaactt tatttttgtt ttcagtcacc tgaaagacat 101101 gacaatccat actccatatt aatgcagcgg cgattctcaa atagagaagg gctttaaaaa 101161 attagaaatc tctgccgggc gcagtggctc atgcctgtaa tctcaacact ttgggaggcc 101221 gagatgggcg gatcatgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc 101281 catctctact aaaaatacaa aaaattagcc aggcgtggtg gtgtgggcgg ctgtagtccc 101341 agctactcgg gaggctgagt caggaaaatg gcatgaacct gggaggcgga gcttgcagtg 101401 agccgagatc gcgccactgc actccagccc gggcgacaga gcgagactct gtctcaaaaa 101461 aaaaaagaaa aaaaaaaaga aaaaaaaaac aactagaagt ccctactcca acttgaaatt 101521 tggatgtatc tccctagagt atgtttcttc tctatgctgc attgcaattt ttctttgttg 101581 ttgatagttg tccagattga ggggaggcag aacaagatgc atctatatgt ttccatctct 101641 ccgaccgatt ctctcccttc cccctctact tgctttcttt ctcttttccc tcttctgttt 101701 acccgattct atttctgatt ccagtatgta acagttccct ctgaagctct ctcaatacca 101761 acaatcctaa ctaatggttt ttaaaagtca aatattaagt actggaggga tagaatgaga 101821 gaataccaag actgataaga tgcaaataat acttttaaca tatttacaat ctaatagaaa 101881 tacaagacat gctcaaataa gttaattatt ttaatatact ctctctgagc ataaaatata 101941 attatatatg ctcattatag acatataaaa aataaatagg tagaggcttt ccatagatgt 102001 gtaatttcac cacttgaaaa ttactatatt tccttataga ctgttttgtg tgtattcact 102061 tatatccatc aagtgactac atttcaaggc actatatgag aaccataaat attgtacaaa 102121 caggatttgc taaatgtcgg tggagagtaa cagtccacgg ggctgatcat ggtcagtttg 102181 tgaggcaggc ctccaaactc cttggggatt gagatgatgg agtagcagag ctcttcaagg 102241 gtatggaggc ctgaaggtac aaagcatgct caggaaattt tggctattgc ggtttgtcta 102301 gagcacttgt tctcaacctt acctgctcat tactaattct actaagtaca gaattaaaag 102361 aagaaaaaaa tctaatgacc atttcctcct gggactaatt agatcaaaat ctttgaaccc 102421 agacattagc gttttaaaaa gctcctcaga tgtactattc agccaggact ggggcaggga 102481 aagctactga actccagcct tgagaatgag aagtagaaca agaggagaac tttaaaagga 102541 tttaggggcc actatatgac tatggagctg aatttagatt tgatttagta ggcaacgcgg 102601 aataatttgt ttctgaacag gagagtgaca caatcaaagt ggaatgatag gaaaattaat 102661 tttgcaagag agagagaatg agttggaagt aaggaactca gaaggcctcc tgggactcag 102721 cagaaagctc tgaggccacc aaatgggtgt ggtggtagtg gaaatggaga agaagggaat 102781 gtaaatgagg ctacacagtg gactgccact gttagccgtg gggttagacc acagcaagag 102841 ttaaaataat tcttcaattt taactccaga agggcctcaa aaagactttt tgtcttgtta 102901 tcatcagcta tatggaaggt agaataaaaa ctagttagga gaaaaggtaa taaatgtggc 102961 ttttgatagg ctgtgattga gttggaaggg cataccagtg aaatcaccaa cacaaagttg 103021 gaagtgtagg aaagcactta ggaggtggct ataagtgaaa atgtgaaaat tctctacatt 103081 aaagggatag atgaagtcac agaagtggat gacataattg agcagggtat gtgtagaggg 103141 aagacgggaa ggttaaggac aaaatcttta catatatctt tcttggagta gaaggaagag 103201 gaaatgttaa aggagatttg attcaatgaa acaagtaggt caggtttcta ttcaaattta 103261 caacagatat aattacaaca gatataattt atttagtttt tttcgcttgg acagcttaat 103321 ttaagtgctt tgtattttct tttcaaaagg tgatggcagt tttggatcag tttaccgagc 103381 agcctatgaa ggagaagaag tggctgtgaa gatttttaat aaacatacat cactcaggct 103441 gttaagacaa gtaagaaatt caataatata attatattaa attgcacatt attaatctac 103501 tggaactctt attttgcata cagttgtgaa aatgcaaaat aatgaccaca tttctactta 103561 agtttaatta tgcaatccta gtttgtcttt tcgttgtgga gtagaaagtt ttgtgttatt 103621 tctcctgttg agaaacaaaa cactgtatct gagaatcctt ataatcgtga tacatagtgt 103681 gttgtaaaac tttttgtaag actcacttac actcctcttt ttactttaga accttgctgt 103741 tcaaaatgtg ctccatggac aagcagccag gcattaccta ggagattgtt agaaatgtag 103801 aaacttggga cttttcagtg ccatattatt gttcctgata ctccacagta gtcagactcc 103861 tagctgcctc cacctgcttc cagaccttga agcctagcaa gctcctgact tcgccttctg 103921 ttttcttcag agtatttatc ttttactttt ctggtctagg gagagaatga tttttatttt 103981 tattgaacat gacttctgtg tgttcagggt gaaagaagaa gtttaatgca tgatctcaca 104041 ttgctaattt gattgaaggt tagaaatctt aaactaaaac tctcactgat aagcttgcac 104101 ctctcttttc tggatttatc cactttaata agaactgcta ttgattactt gctacaaaga 104161 tggagaaagt tagcatgctt atcctatttc ctactccctg tccctgtcca cttcctaaaa 104221 cttaaaattg gttgcattaa ttttcctgat atagtaacaa ttataacttg gaatgatttt 104281 caaaactttt gtttttttag tataccaact ctagacagca tggactgact ccttgctatg 104341 tgagatgagg aaaattaacg ctattctttc tccttttccc atcaccttct caagttcttt 104401 aatttattct attattttta tgtagtgaaa gtttataaca tttatattct ggtctgtact 104461 cataattaaa ttgttcacat tttgtctata gtttggttct gagaacaaaa ccaataaatg 104521 ccatttatat atttttttat ttgtacagaa ccaaaatatt tctacttcta gataaagaaa 104581 tgcaaccttc tgtcactaac ttcttttact aatagaatag taacattcca aatatcaaag 104641 tcaaatggat tctctattgt tatgtattta tcatcaattt ataaaaataa aggcatattt 104701 taatttggtc acatttttac cctgatttaa aaaaaaattt gtttttagag atggagtctc 104761 attaggttga ccaggctggt ctggaactcc tggcctcaag tgatcctctc accttaacct 104821 tctgagtacc agtggtgatt tattttatgt agctttttga ggttttctga ttatatacat 104881 atatttttaa aaaacgtact tcaggaaaag atatatattt tcatcatgac ttcaagtgtt 104941 tctaagttct taatcataca gtttgtataa cagaatctac tttcttcttg aagacattcc 105001 tcattcagca catgacttac tgctctaaac aggagagatg gatttctagg ctgcttgtgc 105061 agtgattaat ctatgagtta gtttcctcgc cctctttgat tactctcaat atttcttgga 105121 ttccatccat tctcttggtt ggattgtcct tagtttttgt tgaagaatat cttcgagtaa 105181 tttttttaag aaaaggtgtt tgtgaggtaa atgttttcag tccttacatg ttaaaaatat 105241 cttagttttg ccctcccatg tggtggatat gtcatcacac tttatttttt aggaatctag 105301 gcttgaaaca attttcttca aaatttgaag aaaattccat tgatttttag tgcccactgt 105361 tgctaatgaa aagtctgcag tcagtcagat gtttgctcct atctaggata cctttaattt 105421 cattttgaaa actgaaaatt tgaccttttg aatttcattt gttttcagtg ttctgaacct 105481 ttacaagtat gtgtttgtgt gtaggttgtt tttcattcca tctaattcat tattttgtga 105541 aaaattgtct tctgtgtatt ctcttctatt atttattatt tcctccctaa catttattaa 105601 tcatttttat tgacaactac tatgtaccag gttaggtgat gggacatatg atatatatat 105661 agtagtaagc taaacccagt caaggctctg cttctctgga gcctatatct agttacttat 105721 gattcattat tgcttatcat tgctccaaga gtatatgtta gatgacaagc cttttgggtc 105781 tatcatccat gtttgagttc cctcttcaag ttttatctat aatttgtgtt acttacttga 105841 ctgtctctta caggtttcta atatttttta gaattgcatc atctattatt tagctttctg 105901 gtgaattttg ttttgataat catattttcc atttccagca attctttcca tcccctctgg 105961 ttgttccttt gtagccatgt ttttggataa aatgtccata ggtgtttctg ttcatgtcaa 106021 ttagaatttt tttttgtatt acttgcatta ttgctttttt ctctgaggtt atttgctctg 106081 tgggttcatc ttgatctttc tcttttatct tgtcagtttt ccaaattgag tagttttggg 106141 tgacttcgta tgaagtaagt actctattga ttgttaaaga aggactgtat tgattattaa 106201 aggtaactag aatgggcatt cttcacattc atgtaggttt gcttgttcaa gttaccactt 106261 tctgaacaag aaggttagac catagacttt taagggctgc atactgcaaa gggatactct 106321 gttctttagg ttacatgggc agggatcact gctgagacca tacctgccaa aggaaggcag 106381 gctttgctct ctagatgctg gacttgaaat tgtttcccct ctgcttagtg ctgcattatt 106441 ttttttgctt cttaatctgc tgcagagtat ctagatcagg gtgtccaatc ttttggcttc 106501 cctgggccgt attggaagaa gaattgcctt gggccatata taaaatacac taacactaat 106561 gatagctgat gagcttaaaa aaattacaaa aaaaaaatct cataatgttt taagaaagtt 106621 tatgaatttg ggttgggcca cattcaaagc catcctgggc cacatgcgac ctgtgggcca 106681 caagttggac aagcttgttc tagatacttc agactctgtt ctacatctct tcatagatca 106741 ataacttgca gcaatgagtt tatcagataa attatgttca cttttcatcc ataaaaaaag 106801 tcatgggagg tactcaccat aggattggtt taatccagtc actctggcca attttcttta 106861 aaattccttt aaccttggta tattggttta attccttcag aatggctttt cccattgtga 106921 taaactggtt ccaggcttca cgagtactta ttccaagtac agaaggaaag agggtacctc 106981 ttttgagtac ttgtatttca aaggaatcaa atggcctaca taataaccca ttaaataaat 107041 agatgtctct cagccccagt gagtcatctg tgcttttctc aacaagcact atgttcaggg 107101 caatgctttg tgctaattgg ctttaggcct ttgttaccaa actgatcact gtggccctga 107161 gggtgctgtt acttagaata ttcccacctt ttctgagagg ctgatgttgg ggtcacttcc 107221 ccctgaacta aagtccaggg gctgcatggg tgagggttga gtaactcagt actctaagga 107281 ggaaaggaag gggaatatac actgttagtt aacagtggtt attcctgtat tctctcttgt 107341 ttggattcta ctggggattt ctttcttttt ttgagtcttt attgacatta ggaatgagag 107401 atagaaacag ggtgagagag gaagtaaaat taaatgtgaa ttcttccatc ttataccaga 107461 actcaactgt atttttggaa tctatatatc cttacttttc ccttgattta ttacaaaaaa 107521 ttcttagtgg ctacactaag taaattttgt aacctttaaa aaatacatag ttataatatt 107581 ttaagtactc tgagtaatag aagatttcat gtgagtacaa aatatcctgg gggcatttta 107641 attattagta acaacagtca cactgtagtt ctagaaccaa attgaacatt ttatatatag 107701 aagcttatct gtgaatcaaa tctgacataa tctcttttaa tgtgtaggta atttcttata 107761 taaattgatt ttttggcctt gctgtcagca tgcatttcaa attttacacc atgtgtttgg 107821 ggagctatgg tggaaattag tacattaaaa atgtctacag agccaattag tgtataagca 107881 gggaggcaaa ctaagggaat ggtggaagaa attaaaaaaa aaggggtgtg cataatgtgc 107941 ttcttctctt aaaaaaaaaa aaagaaaaag acaacaacaa aaagaaagaa aaatgacatt 108001 tgacggtcat tctaccagta gtcatggggc agatctatat tatactgtac tattacgctg 108061 ttttttcttt gcaattagtg agttgctttt ccaggataga aaatttggat tagacctctg 108121 tctgtgccta tgaaaacaag cagagctaac ttaactcctt ctcatcagtt ctaaccaact 108181 gacatgggca ttaaaaaaaa gattttatct actaagcaaa tatgatcaca gtctgaagct 108241 ttgttcttgg aaaatcccct ctcagggtgt tcagcctttc ttcttcagct tgcagaattc 108301 tccatgtttc agtttcctga taaatcagtg ggcgccgcta ctccacatct ttgaagctgg 108361 ttgttaagaa gcagtgcttc tgcagcaatc acagtttaaa gcatgaatca atttaacatc 108421 acacaagcta tacatttcaa cagagttaca gtttcagagt aaagtgcaat atacagtata 108481 aagcgaatct ggaattcaag cccaacaatg tcataaaaga ggctgtgaag tctcacatga 108541 tgtgggccac agagagggtt gttgccattg gatcttagct caaatactac cacatcttcg 108601 gagtggcctg ccatgggcca cccttactaa ggcaatccct ctgtccaagc caaaatgata 108661 tctcggactt tttttgaatt gcagagagat tgttgcagcc agggattgcc ttagtaaggg 108721 tggcccatgg cgggcctctc tgaggatgtg gtaatatttg agataagatc tgaatggcaa 108781 gacttcagcc atgtgaaggg tggagggatt tctagggaaa ggggtcagca caggcaaaga 108841 ccctatgatg ggaagaagct gggcacagct tgggattgaa tgaatgccaa tgtggctgaa 108901 gggtggtgat tgaagaggag ggggagacga gaaggtctgg aaggcctggg gcatgatcag 108961 atcaggtgct gaggccgtgg aagtagatgg gattttcatc taagggaaat gggaagtcat 109021 tagagagttt tcaggagggg atgatatgta tttttttaaa attgagcatt atcctcggta 109081 aacttttgta gtcgttaaac cagagattat aagcaggttt tacctcatat gccagttgca 109141 gctgattagt agtggctata gagaatcctg ggctgagaag gatactgtgg ctaaccagaa 109201 ttcagtagat gagtttgacg tggcctgtta gtatgactac actgtgtgca ctgtttctgc 109261 attaaatgtc tgataaaaac agagccaaag gaaaaataga acttaaaaat ttaattctga 109321 cagtacagtt gacccttgaa aaacataaag gttggggtgc tgaccccttg tgcagtcaca 109381 aatttgcata taacttttga cctccccaaa aaactgaacc actgatagcc cactgttgac 109441 tggaagcttt actgataaca taaacacttg attaacacat gttttatatg ttatctgtat 109501 tatatactcc attcttacca taaagcaagc tagagaaaag agtattttat ttaaaaaatc 109561 ataatgaaga gaaatatatt taatcttcgt taagtagaag tggatcatta taaaggtctt 109621 caccctcatc atcttcacat tgagtgggct gaggaggggg aggtagagga aaggttggtc 109681 ttgctgtctc aagagtggca gaggtggaag aaaatttatg tatatgtgga ctcatgcact 109741 tcaatcccat gttgtccata ggtcaactgt agtttcaaaa ccagcttttt attactgaaa 109801 atacgggaaa aaaaactcag agaagaaatg gaaagtttgc tatgatccag tcatacagag 109861 aaatccatgt tcagcctgtt gatgcacttt aaagaaggag atacgtgggt aaaacctgat 109921 gttgaattac tcttacatga ttttggactt ttgcaggagc ttgtggtgct ttgccacctc 109981 caccacccca gtttgatatc tttgctggca gctgggattc gtccccggat gttggtgatg 110041 gagttagcct ccaagggttc cttggatcgc ctgcttcagc aggacaaagc cagcctcact 110101 agaaccctac agcacaggat tgcactccac gtagctgatg gtttgaggta agtaggtcat 110161 gttgttttct attcagtgca tgacaagtgt gatccagact tgctctcagg ttctgagaac 110221 acttcccagt aacactgtgc cccagtaaca atttataaac aatttggatg aaaactacca 110281 tttccctgat caaattttgt aatttcagaa aataagagta tggaaaccat gcagaacctc 110341 atagcaagta gtaatagact ttgaacccac aagttctgct ctagaaccca tcatcttaac 110401 cctgtactga tctgccttct ataaaaatgt ataagttagg cttcacagta tcaaagtaag 110461 tgtcaattac atgattccaa tgaggaaaga tgagtccata cttctcaagg ggactagagt 110521 gattcatgtt ggattcttcg gcatgaccat ctcacatgtc tcagaggcac acctaaccct 110581 gcatccagag caagctttgg agagggagca cactggagtg gaaaggctgt ggtctttgaa 110641 gacaaaaggc ctgggattca tcactattcc acacatttag taactgtgat tttatatctc 110701 tgattcccat tttttaaata gtctgtgaac catgactaat atttaatgca taaaattatg 110761 atgacttctg taataattgg agacattcca gatgaaactc ttgatgtccc ctctgccatt 110821 gctccccaac cccagtcacc ctgttacacc tgagagtcac cttacattcc tttcttcctc 110881 tctcatttca cagctaatcc ttcagcaaat cttttcagct ctgccaccaa aatatatctt 110941 aatgcttcta acaatttctc tcactaacgt ctaaatctga gccagtatca tctctcattg 111001 cctactggtc ccctgcttct acctctgtct catgatagtc ccattcctca cccagcctct 111061 ggagtgattt ttctaacatg aaagttggat caggacttgt tcctgttatt acccctcccc 111121 tgccttattt cttgggtaca gtgctcagcc actcccatcc ctgaggttcc ttgcagatac 111181 cagaggcttt atatctgctg ttgatttcac tcaggaatgt ctgactccca gatgtgctct 111241 ctacttatta taaaggatta tctgaatctt tctgaatcct ttcatttagg actctcagca 111301 gagaggatgt ccgcaacgac cctttgtctc tccagcccct ataggactat tgctgcctag 111361 gattctttat gttttcattt tttaaaaact tatttattgt ctgtcttgcc atcagaatct 111421 aagtaccatg aaagaaggga cttttcgtct tgtttgccat tgtatctcta gctcctaaaa 111481 tagtaagcct tcagaattac tgtgttgaca gtaggggaag ggggagaaag gaggaaagaa 111541 ggaaaacagt gcctggggca tagaagccaa gcagtgtatg caactttcct tctcttcttt 111601 ctcttctgaa atgctatgaa tatgcctttt aggtagtatc cagaaatgtt ccttcctgaa 111661 agggtccaga aactactgaa aactgtacag attatgaaat gaaacagggt gcagggattt 111721 ggatttgagt tgatgtttct gcttttgaac accaggggga atcttgggtt acattaatct 111781 aggtaaagtg cagaatagtc tcctgtattt cagtgccctc tttccttcat ttaactaact 111841 ctaggttcta gtttttccct aattcttcca caaatcccca aagtgtttat ttataaagtg 111901 aagaattgct attttttaac actgttcgaa acaccttatc tctaaaatga cttattctag 111961 ttctctgaaa ccttacttta aataacaaat ccagcagttt ctgatgaagt aaatgaaatg 112021 tcagcatatt ttaaaataat ttgcctaatt tgttcttagc ataatgccag aaaagctttc 112081 tggattttgt atcacaaaag gctagtagat ttcagtagct atcaatcttc taccagcact 112141 aagtatattt taaaaactca gcattaaggt ttatttttcc aagtatgttt cagcacagga 112201 aataaaatca tgctcctttg gagtccctta aatgctggag ctgtttagag tgacatacaa 112261 gaactttctt cacgttacat gctctctctt cctccatctt gcttttaact gttagcttac 112321 ttctccaatt caatccactt cgtttgaact ctttatcata attctataaa acttatgaaa 112381 atacagtcaa ctgcattttc tgtatgtttc tgtgtttcaa tatcttcaaa atggaatgta 112441 ctgccttggt acatcaccca ctatgaatct gttatttctg ttatatccca cagttgccag 112501 gccaggatac ttgtcccatc caggccaaac accttccccc gaaagcaagt atgcatttgt 112561 ccaccaggtc cttgactcta ttttacatta tctttttagt caattcattt atttttatgc 112621 cactcctgct gtcttggttc agtatgtcca gggaattatc agaatttctt ttctaaaata 112681 aaaatctgtt tatgcttgca attccttgac agttctcaat tatctgcaaa gtgcatccaa 112741 acttcttggc atagcatcaa agatctttct gtatgcctct tgcttccctt tgcggcccct 112801 gccaccccac tgcccacact gcattctagc cgtgatgaca ggcttgaatt ttcagttatg 112861 ctcatgtctg tccatcattg tatttgttat tcctctcttt ccaccaagtt gtctgcctag 112921 agagctcatt ttccttaaga atttcttcac aaaccatctc tactatgaag ctcaagtgtg 112981 tcatgaagtg ttagcttctc caacttgtgt ttcttgcaga cactctgtgc aagacattga 113041 cttaggtgct aaagagggaa agctagatat tatattgttc ttgaggttga aagcttacag 113101 tctagtagga gagtcaactt tgctgtcttt acctcagtgt ttttctccct ctgtgcttcc 113161 ctagcacgtg gtacttacat atttctggaa tcttgattaa acacctgttt gaggactgtc 113221 tgagcacaat ccttctggat tgtgacaccc tcaagggagc agagatacaa agatggcttt 113281 gtatactaaa tgactggccc tcatagatac ctagtacata tttgtcaaat aaatgaatgc 113341 attctatttt tggaataatt ctattcagaa tcagataaag tttactttaa gctatgaaga 113401 aagaagtctc ttagcaactc ttacaataat cacaatcaaa gaatgactgt ttaacttaat 113461 ataaaccagt ttgttttaat aaaatatttg acaatagtca tggttacaca atgcataaat 113521 tatggctaaa ttattatcag gaaggaaaaa tctttactta ttatttcaaa agctattttg 113581 ctagtctatt aaaagctatt agaactgcac ttcttaagat taaattctat aattgaacat 113641 tttaactaac caagatatta tctctttgcc actgacatta tttcaaatta agcttaacta 113701 tttcttttta gcctttggaa agtattctga aagagtctgt gttctataaa tatacttaaa 113761 gaggcatgtc ttataaagga tttggatact attcaatgat gtatgacttg gctttagctt 113821 ttttattctt aatctctcag cttttctctt cagcagggga agagtaccta atggcctttc 113881 agtaatccct tggtaaattt ttctttcaag cccattactt actgtgaagg tcaacttcat 113941 tagtgtattt atcttatttt tttcagccca aaataggtat attgaaatga atgggcctaa 114001 tgtcaaatgt cccgactaca tcctggaaga gagagaatct tcagctgtat tagttgatgc 114061 agttaaataa tatgtactct ccaggccctc atacaattga aagttcaggg tatcgttgct 114121 gctctgcttc taatccttcc agaagtgatt ggtgctaggt gatggagtaa ctattaattg 114181 atataatgtg agccaaaacc aacagtcacg aataagcaaa ggatttaaat ttaactccat 114241 taagtcttgt gagaaattat tttcaacata ggttataaca tacctgtgac atcacatgaa 114301 atgctgtagt caatttgaca tcatggggca gagaagacag agttggaaat cagaatttta 114361 tagacatcta atgtgataat aacattagta gctgagatgc ggtaagctct ttgaccatgt 114421 ttccagaatg gataagacct ggttgagatg aaaactttac actgtttttt tatattaact 114481 atcttttact ctttgcctga aatgtccaac tctagttgct cgtgattgcg tgggtcagtc 114541 tccagaaggt tggactttaa tattacccgt catcttttcc aagacaaaat tgtattcatt 114601 ctaactctta gccccaaatt ttctttttta accttaatat ctaacatgat taggtttatg 114661 gtaaattata tactcaaaca gaagaagaga ctaatagcaa gcaaaagtct tatattttca 114721 tttgttttca tccaaaaagt agaaaatatt ttccaaacat tgggaaacat tttagtcaga 114781 aaaataaata tcaatgataa atagaataga gaaaaatttt aaagctgagc taaacctcta 114841 tgtggtttta ggaaaatcaa aactattaaa taaatggcaa gtacaacaaa atcccatcaa 114901 ttcttattta acatacttac attttgaaat agttaaaata ttcatatgat cattgagaga 114961 attcagaatt gcctttaagt aattgttcac atatacaaaa gaaaagtctc caaaaattgg 115021 gtctttgcct gagatagatt tgtcttaaaa ttgaaatcat tcacttatca gatttgaccc 115081 ttttttaaag cataactttg ctgtgtaata ttagacttat atgttttgat ttccttctac 115141 aatatctctt aactttaagg gacaaagtga gcacagaatt tttgatgctt gacatagtgg 115201 acatttatat ttaaggaaat taggacaaaa attattataa tgtaatcaca tttgaataag 115261 atttcctgtg cattttctgg cagatacctc cactcagcca tgattatata ccgagacctg 115321 aaaccccaca atgtgctgct tttcacactg tatcccaatg ctgccatcat tgcaaagatt 115381 gctgactacg gcattgctca gtactgctgt agaatgggga taaaaacatc agagggcaca 115441 ccaggtaggt gatcaggtct gtctcataat tctatcttca ggatggataa ccactgacct 115501 cagatgtgag ttcagaagag tcaaaaggaa aacagagtct atcacattgt gaacagaggt 115561 ttattttgtg aaaaaatgca agcatcacat tgtgattttt atcattgtat tttgtaggaa 115621 aaaaacaatt gatgtaattt ttcagggcaa aaactgaata aaaagaagag aatgtttgat 115681 atcaagttat atgttttaaa gttagatttg tagattcttt agatactcta gaggtcataa 115741 aaagtaacag caaaaacttt agtctaggta ttgttggcac ttgtgaggca aatcaaattc 115801 aggtccacaa attctttttc ataattctga aacccaaaga actctgaaaa tcccaagatt 115861 ttttaaaaaa tgactaattt ggtgtcaaaa cctaagcaag ctgacttgtt gcttattaca 115921 atctttattt ctcatgctca gtgtgaatat gcatacattt tgctgcagaa atatatacat 115981 gtttgagtac agggggctgg ccgtgaccct actgagggtt tctgtacaca tcactgtcta 116041 ccctgtggaa tcttacctcc ctttcttagt tcccaatcct gaaaagcagt tatggggcca 116101 gtgctctgta cagacatgtt gtctcagaca tcagtttgag caggaagtaa atcatttagg 116161 ggttggcatt tgtttggagt gtggggaaca ctctatcttt agggaaactt tatatagtta 116221 gttatttgta agtaaaatta caggtggcta tacatcatct tgctgattgc aactcaatta 116281 aatcaccgtg cctggcacag aagaaaatat gctacaggat atctcactag ggaaaaggtt 116341 ctagttcgtt tcctgcgcac tcaacttttg tacttagata agcaaatggc cccagattcc 116401 aatgcctggt tttatttttg ctccaaatac atatatactc ttttgttttg gatagttaca 116461 ttttagaagt agactgtgta ttctcataaa cacttcaaag tgtatgttct ggctgagagt 116521 gtctctgtgt tgttcaataa taataagact aattatcatt ttttgagtac ctgctgtgcg 116581 tcaggcccag tgccacgtat attagagaca agatctctta tcctcatgcc agggctggaa 116641 gttagctatt agtttctcat ttgccaaatg agaaaactga ggctcaggga gattatgtaa 116701 cttgcagaat atcactcagt aattggccaa gataagaatt cagtctaaat gagaaccaga 116761 tccagagata tttggcttta aattctatag tctctcctaa accatatgca actctaacat 116821 gaagaagctt atttaatctt cactattaaa aaagtcaaaa caaaacaaca gagccatgaa 116881 tagcaaatat tgtcaatgag aggtttggaa aaacagtctt aaaggatgaa attccataga 116941 cctgatatat ttccacctgg aaaaagtggg catgggacag tgattttctc ttgaaagatc 117001 tgctcatttt tgtcatggga catgaaggtg gactggacca ctcagtttct tctttctgca 117061 tctcccaacc cagtctttct gttcatgggg tgaaaatctg ttgttgaagc cttgtctgct 117121 taattggaca gtggatctct cgggtccctg tgggctgtgc gcttgtactt gagctctgct 117181 tcttcactct gtggtctagg ccagctagca gccagctgag ttcaccttgg ttcagactca 117241 tggcctttca ttttcagtat ctgacttcct ggttttgctg aaaacctgtc taaaatgtaa 117301 tatccatctg attcttcata ccaagccaca caattcttcc tgatcccttt taatctccaa 117361 tattgaatgg tggtaacata aatatggaga cagatcatgt cagaaaccca gggcctaatc 117421 ttttcttttc tgcctactct tctcacaggc tgcttagtac tttgtaagct tttttttttt 117481 ttctggctgt aacctagatt ttctctttat cattactcta tttattattg ttagagcact 117541 tctgattatc tcagccctaa actctgcctc caattttaaa taacaataac tcccactcct 117601 gctaatactg ctactactac taccatcacc aaactttttc ttccccaaag cagttctgtt 117661 tgggaaggaa acagttccct ctcatacaat ttcagttatc ttcttgtctt ttccgtttaa 117721 tgaatcttcc tgttaatgtt acatctttta acatggaaac ttctagagaa acaaaagacg 117781 atggatttgt taaacctttt gggtgtattt ttatactaac tcttactgca gcgtgtgcat 117841 tatgagtgta ggtccattac ggctgtatta ggagcagaac cttccagagc atgagcgatg 117901 tgctgggctt gtgcttagct ctatccatga gttaagtatc tcaatcctta ggaccctctg 117961 acatatgtgc tattattatt tctagtctat agatacagag actaaagttt agagaatata 118021 aaaaaacatt tacaaggtcc tatgggacaa aaactgtagg acaaaatgca aacccaagca 118081 gcctgagagc agagctcctg gtccagcact gtgatagctg gggacgcaga gacagaaaca 118141 atgcaattat tgacagggac catggtgctg tgtctgtcca cattttgaag ataattatgg 118201 tttggatatt ttcaccttta aataacttgg agagtttcaa cattaactca gtcagatgga 118261 tacatttata tcatatcctg ctgggagtga cagttaattc tgggtcctat ggcaattgca 118321 cttttgactg agatgaatgc tgactgatgg ctttaacatt taactaatgc gatagtattt 118381 aacacaccca tataaatact atagtcttcg ggtaaataaa atgttaccgg ctggacatac 118441 atgaatatct gatggagatt atggaacata ctctactcat acttctctga aagtaaaaaa 118501 taaaagatat gtttcagtac acaatgtgat atgtactcag acttaattca taaatttctc 118561 ttatccttca tccgtggatc ttttctttat ttacttattg cgtttgttaa aatgcaggct 118621 tctctgaaaa attattttta aaaatagttt ttagacaatg aatcatattt tctcaagtat 118681 tttaacattg taatcattat gataattatc caaggggaaa ttatacttat tttttattca 118741 tttattcatt catttggcaa caatacattg aacatttact aagcatcaaa ctggctctac 118801 cacttaatag tggcatgatg ttcatcaaga aattgttagt gcaatcaaga acactagaaa 118861 ttcattggat gaatttaaag aagcttttag aagggtatta tattataatt gaggcacttt 118921 atgaatatat aaataatatt atgttttcat gctagagatc atgccaatga agatatttac 118981 tttgaaaagg agaagattag aagtttaaaa gcatttccat attgaagtaa atattcattt 119041 ccatatcttc acagttatct ttctctgagt tctctgactc attgtgaaaa aaaatcccaa 119101 ccttcttcac agctctacca tcttcggatt gttgcctaga ggggtaaaaa ctattgtaaa 119161 aggatgtgtg cactggatga gaatttagaa ttagacgaaa tgacccctag agtcttttcc 119221 tgctttaaga gcctgtgatt ccaaattcta acagtacatt tatcaagaaa aaatatgctg 119281 aacatttaaa tagtttttga atagtaccta gatataatag atacctaata aatgtgctca 119341 atgaataata aataactggt taagatttaa ataagcctcc aaaatctctt ccacattcta 119401 agaagggaag cataaaggtt gttaatgaac tagtgactgt gtgggtagct cattattttt 119461 aagtactctt gactttgctg ttcattatct gtgtggcctt aggaaaatac acacatttct 119521 gaaaggatta tgtcgtttgt aaaatagaaa gtccttatct gtctaccaca gatgattctt 119581 atgcaaatca aatgaaatgt tcaataaggt gtctgtaaaa tagtagagag agatgaatta 119641 ggagctattg tgatttgttt acattatgtc acaggtgcac tttattaggg atatgtttta 119701 tcttaattac acaattcttt aacttagatt ttgagaatta tattatggtt atataggaaa 119761 atgcccttat tctaaggaaa tgtataatat atttaggtct gaaacattgt atctgtaaca 119821 atatagtatg taaattatgc taattcacat gataattata tgtaattata ttaatatatt 119881 actatgtata caatatattt acatgcatat atgtggggaa atgttatcag ttagtgtagt 119941 aggggttatc atactcaaat tcgatgtctc catccttcca actcttcatg cttttccagc 120001 atggtgagga ctgctgagct ccatcttttg ctggtagtct ctctgtcaaa tagaactgtt 120061 tccaaattca gtcatttgct ccttgaaggc tatgaattca tacttcgtta tatttttctg 120121 gctgcatatt taaattactt taacaatcat ataagttcat tgtaaaaatt ttggaaataa 120181 aaaggaagat aaaatgcaca gataatttta gcaaatgaaa taataattat attgggatgt 120241 atttcttcct agattttaat tatgtacatt cccatcaact ttttattttg aaaatgttta 120301 agcctaaaga acagttgaaa gagtagtaca ggctgggtgc agtggctcct gcctgtaatc 120361 ccaacacttt gggaggccga ggtgggtgga tcacttgagg tcaggagttc aagaccagcc 120421 tgaccaacat ggtgaaaccc tgtcactact aaaaatacaa aaatcagctg ggcacggtgg 120481 tgggcacctg taatcccagc tactcgggag gctgaggcag gagaatcgct tgaacccagg 120541 aggcagaggt tgcagtaagc caagatcaca cccctgcact ccagcctggg caagagagtg 120601 agactccatc tcaaaaaaaa aaaaaaaaaa aaaaagaaag aaagagtagt acaatataca 120661 ttcatactca tatacccgac gcataaattc attgattatt tactttttgc cctacttgtt 120721 ctttcttgct ctctttgcgt atgaatgaat cattgaaatt aagttgtaga catcatgcca 120781 tatcacctct gaacagtgtg tatatttctt tagaataagg atgtttactt acataatcat 120841 aataccatta tcacagctaa gaaaattaat tcagttgatt ttttccacat atttgataac 120901 tttctgtcta tccacgatta tgtcttacat attcttttaa tttatgtcat agcatatcat 120961 cttagaaagt gatccctaag ttactgcatg gtatacattg tttaaccatt tccctttgtg 121021 attggatgtc tttaggttga ttatattttt attattatca caaatgttga aatcactctt 121081 tttttctgaa gaatttaaaa gtaatttatc tgtcttatgg aataaaatat ttatttcccc 121141 ttaaaagaat ttcaggcatg aacccaagag agaaggcttt tttttttgtt ttagttgttg 121201 tttttatttt tattttttat tttttgggta gaaggagcag agagacaagt tcaggaaata 121261 atgagagtgt tagaattttg ttcaggttaa agtgagttgg agtgaagttt agaaatctcc 121321 tttctactca tctctcctgt ttttaaaaca ctgtcctgga aatagttaat attaggaacg 121381 agaaaaatgg tataggtttt cctagtacac tttatttctt aattatgaaa ttctacttaa 121441 taacttacca ttgaatgttt atccttatta tcattcaagg taattttatt gaagattgaa 121501 gatatttata ataaagattg aaggatttta ttgtcctgtg tggtcaacct tggggggtga 121561 gatgttatga gacaggacaa ttaattgact tgatcaaggt accttgttat aaaaataaca 121621 cagcctggtt tagaacatct cttcctgact ctcttatttg gcatatagcc taagtgtatg 121681 cctccttgga tgtatgagcc ctgatgttgg tcatatttat tattttatct gcttactttc 121741 agggtttcgt gcacctgaag ttgccagagg aaatgtcatt tataaccaac aggctgatgt 121801 ttattcattt ggtttactac tctatgacat tttgacaact ggaggtagaa tagtagaggg 121861 tttgaagttt ccaaatgagt ttgatgaatt agaaatacaa ggaaaattac ctggtaagtt 121921 ctgttttctc tacaatgaag attttttttc ttaatatcag cagcttcatt tttatttaat 121981 tgtagttgta tgcttaattc cttaaacaga tgatcatttt ttttgtttag tgcataaata 122041 ttcttaaatc ttgtgatata ttaataaaaa tcacctgaaa aaggtagcag ttttaggctt 122101 tttaaaaaat ccgcaattaa tattggtgta gttaatatta tatttagaaa catagagaag 122161 gaaattgctg ttagaactcc acatttggtg atttttaatt ttcataaaga attactgtgt 122221 actcattatc ctggaatgtt ttcgttttct tggagtgaaa taatttacat gcaggaatgg 122281 aagactgaat gatctataat aataattttt cataagaatc ggtaaatgtg tatttaatgt 122341 tatcaaagct catttggaat ggttgtctca tgctttcaag aaattagagg actttgtaat 122401 tcattcctta accattactt tagttctcac cacaaaataa cattttaagt ttatttagct 122461 ctttctcata ttttctgctt tccctttcat ttaaaaaata cttttgagtg tacacaatgt 122521 gccatgtaca ggaaatagag ctttatcttt tttgggtata acttcaagat catggcaaaa 122581 gaaaacttat tattaattgg ataaacctta gatataatct aggttatttc ccttatttta 122641 ctagttttct agtgaaaata ttcaggtctc tgctgggtac agtggcttac gcctgtaatc 122701 ccagcacttt gggaggccca ggcaggcaga tcacttgagg ccaggagctg gagaccagcc 122761 tggccaacat ggtgaaaccc tgtctctact aaaaatacaa aaagtagctg ggcatggtgg 122821 catgtgcgtg tagtcccagc taccaaggag gctgaggcac gagaatagct tgaacctggg 122881 aggttgcagt gagccgagat tgcgccactg cactccagcc taagcaacaa agtgagactc 122941 catcttaaaa aaaaaaattc agttctgtgt tctgcatcaa ccagaataag ctacgcctct 123001 tataaaaaac aaatgtgcac aaaccatctg tgaggacata aggattaaat gcttgcttac 123061 tttgagtatt aaaataaaaa gtagaagctt tattatatga gtaaaagtgt ttccaaagtc 123121 tatttgaaat gcaggtacag aatgaaaatc tgttatttta ttaaatcgtt atttgggtct 123181 ctttttattc cataaaaaaa aaatcttttc cacatctctt agtggagatc aagttaacaa 123241 aattagcttt aattttgtga caagtaaatt tacataaata taggattatg gagataatat 123301 ttttctttgc aatgtctgga ccttttataa acattgagag gaaatataac cattcttact 123361 tatttagtat gctagcatga tgttttttaa tgttttagat ccagttaaag aatatggttg 123421 tgccccatgg cctatggttg agaaattaat taaacagtgt ttgaaagaaa atcctcaaga 123481 aaggcctact tctgcccagg tattcttaaa gttttgttaa tattttgtac agaacatcat 123541 ttgcatatat gcatatatat ataatcttca aatatatata cttaaacaca taaacacaca 123601 gagacagaat taaaaatagt tataaggcaa acctcctata attttcacca tcccaggcac 123661 aaaaaaagga cattgccaaa acctcacatg ctcccatatg cctgtctcct cttctcttct 123721 ttgaaatgcc tgcccattat tttgcccatt tttctgttga ctcttttgcc tacaaatcaa 123781 atcagaaaaa aatttttata tataacatac taatcctttc ttggttttat atgttgcaat 123841 ttctttttcg agctggtggc ttgcttttta aatttcttgt agggtgcccg ttaatgaatg 123901 gaatttctta attttaatat atataaattt aacaattctt ctttttcctt cttttcctgg 123961 ttattcctat ttggtcctgt tgtagagccc atctttcttt tagagctaca aaaatattta 124021 cctgcaattt cttcaaacat tttaaaagtt tgcttttgac aagattttaa tccattggaa 124081 ttgacatttt tatctgtatc ccattccttt ttttaatgtg gaagactaat tttttcaggt 124141 tgatttacta aatggcttct ccccttttgt cccatagatc tgatgtgtcc attttgtaat 124201 ttattaaaga taatgtgcat atccacgtgt acactgtctt ccactgatca actcatttat 124261 ttttcctcca gtattaccct gtcttaattc ctgtagcttt ataatgatct cctctcccta 124321 tttatttttc tcatccagga atattttaac cagtcttagg cttagctttt ctatataaaa 124381 tttagaatca tgctgtcaaa tcttatgaaa aaccacattg tcgtttggat tggtcttgga 124441 ttgaatatgt tgacaatctg gagatgatat ccttatgata ttaagtcttt gtattctttt 124501 tcttatttat ttatttattt agaggcaggg tctcactctg tcacccaggc tggagtgctg 124561 gagtgcagtg gcaggatcac agctcaatgc agctttgacc tcctgggctc aagtgatcct 124621 gtcgcctcag tctccatcag ccaattgtgt gccaccagac ctggttaact tttcctttaa 124681 tttttttgta gagaaagggt ctcactctgt tgcccaggct ggtcttgaac tcctggcctc 124741 aagcaatcct cccgcctagg cctcccaaag tgcatgagca actgcacctg gccaagtctt 124801 cctattctgg aatggcatga attctctctg tttacttagg tcttctcaag cgtctttcaa 124861 taaagttttg taatgctaca tacaggtctt gcatatcttt tgctggattt tcttgctgca 124921 aattgcttta aatgcagttc atggcttgag atttgttttt taactgccca gttaacgtaa 124981 atctggtcta tgaattgtat tgacaacaag cttgtgagta gcaatttttc agcaaccttt 125041 tgctccctct ccccgctccc tgcacagtcc cagaacaact ttccttatat accaatgaag 125101 gtggaaatgt ggacaggttg atttctcctt cttgtttatg ctgaatatgc agtcctttgg 125161 ggtcccagct ttataggtac agtcccttta ctaagactgt ctatcttggg aaggccctag 125221 actccaactt ctctcccatg ggccccacaa agcatccaag agtatacatt tatattatct 125281 cactttgtct tggcagacaa atgtcttcag ggcaagtctg gctttagggg tccactgacc 125341 tctctggttt ctgtctctca ctgtgatttc ggcctgataa ttccttatgg tgtcagatct 125401 tcaatgtttt taatacattg tttttaaaaa aatcatttag cattttaaat tgtttttagt 125461 tggaagattt gcccaagtaa ccttgtccac catattatct gaaagagaac tcttgtcatg 125521 ttttacactg atacacattt taataaatgt gggttatctt tatgttgtga gctcttgatt 125581 tggtattata attaattgga aaagttttaa ctttaagtat tctgatcaaa atagtcaagt 125641 acaactataa tgactttatc aaatattaca taatttttct tctacttggt ttacttgttt 125701 tttaatttag gttaccatca atgttagtca catgaacttt tatatttatg tccacagtaa 125761 aaatttttca tagcttgttg tttttctttc ttgttttttc attttcaatt atacttcacc 125821 tatacttaac agaatactta acaaatatgt atatatatga caatattaaa aagcttagac 125881 atacttattt tatgtttaaa atataacata tactaggcaa gacaggaaaa ctcatcactt 125941 ttatgaaatg aggcacagac agagtaatgg gctttcttgg tgtctcctga gtggcgggca 126001 ggtggccatg tcacagctct aatcccagtt tcctgacttc tggttctgtt ttcttacggt 126061 gccttcacac tgtctctcca gatcaaaaac agaatctaga gatgacttct aaattttgtt 126121 accaaagact gaaattcctg ttcctttcaa ctactagaag ctcaactaaa ttgttggccc 126181 aagggttttt ctctcactgg cggtggctct gcaatataga attgcatgca gagtacctcc 126241 tgactccgct aaaatcctgt ttaattgacc cttgagattt tcttttcaag ttaaaaaaaa 126301 tactacacat accaaagagt atcaagcaca gtttaaaaat acatatttgt cttttcatgt 126361 aattttattt gtagtttaga ttactaatct tggtgatcta gttgggttcc agtttaacag 126421 ttttaggttt tgcttgacag agctataaca cttcagtcta tatttgattt ttcaagggaa 126481 atgagttaac tcgataagta ctgttttgtt atctttaaac tttctcaggt ctttgacatt 126541 ttgaattcag ctgaattagt ctgtctgacg agacgcattt tattacctaa aaacgtaatt 126601 gttgaatgca tggttgctac acatcacaac agcaggaatg caagcatttg gctgggctgt 126661 gggcacaccg acagaggaca gctctcattt cttgacttaa atactgaagg atacacttct 126721 gaggtaaatc caaatgctct ttaaatcttt cataatttaa agcatatacc atttggaaag 126781 ttacttagga ataaattaaa taagagccaa tgtaggatta ttattcaatt agccttctgt 126841 tagaacaaga gtattcaaga gcaaatgtgt tttgctttag aatcacagca tatgtcttag 126901 ctcagggtcc ctagaaacag agcctgagat gggttatctt gcacaagtga tttaaggaaa 126961 gagaatctta gagagaagta gaggaagcca gaaagggcag aggaaaaagc tcagcagaaa 127021 tgtggtttct gaagaggtcc agcctcagtc tctgcccaca aggagctctg gaatatgaat 127081 aggagcacag aattttccca ctgccaggca agggagccag tctttcattc tcacatattg 127141 ttcagtcatt ggctgcaatc tgctgggagg tgggggtagt gtaactttca aggcatttct 127201 gggcaagctg cctcctgtca tctgagggta ttctgtgata aatagcacat ctctgaacta 127261 tagtagccaa cactcagggt agctggggat ggcgtacctg atggataaag gagatctgga 127321 catggctcct aaaagtggat caatacattg tgttggcaaa tataagataa gtttctagac 127381 ttcaaagaca acctagtatt ttgactgctg cctgaagata aatattgtgc ctcaacatta 127441 gttctgaggt taaacaatct ttttttttta attgatcatt cttgggtgtt tctcgcagag 127501 ggggatttgg cagggtcata ggacaataat ggagggaagg tcagcagata aacaagtgaa 127561 caaaggtctc tggttttcct aggcagagga ccctgcggcc ttccgtagtg tttgtgtccc 127621 tgggtacttg agattaggga gtagtgatga ctcttaacga gcatgctgcc ttcaagcatc 127681 tgtttaacaa agcacatctt gcaccgccct taatccattt aaccctgagt ggacacagca 127741 catgtttcag agagcaccgg gttgggggca aggtcataga tcaacagcat cccaaggcag 127801 aagaattttt cttagtacag aacaaaatgg agtctcctat gtctacttct ttctacacag 127861 acacagcaac aatctgattt ctgtatcttt tccccacatt tccccctttt ctactcgaca 127921 aaaccgccaa cgtcatcatg gcctgttctc aatgagctgc tgggtacacc tcccagacgg 127981 ggtggcagcc gggcagaggg gctcctcact tcccagaagg ggcggccggg cagaggcgcc 128041 ccccacctcc cagacggggc ggcggccggg cgggggctgc cccccacctc ccggatgggg 128101 tggctgccca gcggagacgc tcctcacttc ccagacgggg cggctgctgg gcggaggggc 128161 tcctcacttc tccgacgggg cggctgctgg gcggaggggc tcctcacttc tcagacgggg 128221 cagctgccag gcaaaggggc tcctcacttc tcagacgggg cggctgccgg gcagagggac 128281 tcctcacttc tcagacaggg cggccaggca gagatgctcc tcacctccca gacagggttg 128341 cggccgggca taggctctcc tcacatccca gacggggcgg cagggcagag gcgctcccca 128401 catctcagac aatgggcggc cgggcagagc cgctcatcac ttcctagacg ggatggcggc 128461 cgggaagagg cgctcctcat ttcccagact gggcagccgg gcagaggggg ctcctcacat 128521 cccagacgat gggcggccag gcagagacgc tcctcacttc ccagacgggg tggcggccgg 128581 gcagaggctg caatctcggc actttgggag gccaaggcag gcggctggga ggtggaggtt 128641 gtaggtagcc aagatcacgc cactgcactc cagcctgggc aactttgagc actgagtgaa 128701 cgagactccg tctgcaatcc cggcacctcg ggaggccgag gctggcagat cactcgcggt 128761 taggagctgg agaccagccc ggccaacaca gcgaaacccc gtctccacca aaaaaatatg 128821 aaaaccagtc aggcgtggcg gcgcacgcct gcaatggcag gcactctgca ggctgaggca 128881 ggagaatcag gcagggaggt tgcagtgagc cgagatggca gcagtacagc ccagcttcgg 128941 ctcggcatca gagggagacg gtggaaagag agggagaggg agaccatggg gagagggaga 129001 cggagaggga gagggagagg aacaatcttc ttatatggtt tgaaggaatg agaattcaca 129061 ctgaaaaata atttttaatt ttagtttcag atgtcatctt gataggcaaa acttgtctgc 129121 caattaactc atttattgct gaaaattaaa taaaattggc attgttttta aaagtaatgc 129181 aagaaagcaa aaagagttat gttgataaca gaatccttta ttctgtacaa gttctagttg 129241 cttaagctta aatcaaatcc tgctaagtat attttctttt cttaacagga agttgctgat 129301 agtagaatat tgtgcttagc cttggtgcat cttcctgttg aaaaggaaag ctggattgtg 129361 tctgggacac agtctggtac tctcctggtc atcaataccg aagatgggaa aaagagacat 129421 accctagaaa agatgactga ttctgtcact tgtttgtatt gcaattcctt ttccaagcaa 129481 aggtatggta gtgaatttga tcaatgggga aattacagat cttttaaacg actgaattgt 129541 gtgcataatt gttattgcat cagcaaagat tgttcatttt tagcctattt tcattggttt 129601 gcatatatta aagggaattg tggaaggtca cagagatatt tgttgttttt ctgaatacag 129661 atctagctga gacatttata aaataagtca accatttatt caggcctacc agccctgctc 129721 ctggtattac ctcaactgtg gctctatctc tttacttctc ctcagatcaa tgaatctttg 129781 tagggcctct tcaaggataa attctcattc attcattctt tgaaaaaaaa aaaatatata 129841 tatatatata tatgaaaccc attgtgtgcc aggcttaaac ataccagtta tctaactacc 129901 aaattaagaa aaaaattaaa taaatgaatt aataaattct taataggtga aaatgactta 129961 gctcttatca attgcagggt tcttgtccca aagaaatata tctacatagc aaaatttcag 130021 gtgtgagttg taggttggtg actgtaatat ttggggcagg atgatttcca ggaggcatta 130081 agattatacc ctatatattt ctctggttta agttagtatt ggaaaaaaag tactagaaaa 130141 atgtgaagcc tgttttttgt acctgaaata tcaactccac tggcagtttc ggagttgaaa 130201 ttatttgaat atggtcaaag aaaaatttca atggatggaa ttgggcaagg acgactttat 130261 tcaagcctat cacagcaggg gagagagatc agactgaact aaactccact gaaacaaaag 130321 gtgggagagt tttaagcgca ggggtgagct aatggaaacg tactggagca ccttgttgga 130381 aggaagtggg agcagttgtc aatgtgatta ggccatctgt gtttgctaat tgtcccttat 130441 tgaaggtagg ctcctactct cccacagaca ctggggaatt ccttccttcc ttccatccct 130501 ccctccctcc ctccctccct tccttccttc cttcttcttt tttttttttt tgaaggagtt 130561 ttgctcttgt tgcccaagct ggagtgcaat ggcatgatct tggctcactg caacttccac 130621 ctcctaagtt caagcgattc tctagcctca gcctcctgag tagctgggat tacaggcgtg 130681 caccaccaaa cctggctaat tttttacatt tttagtagaa acgggatttc accatgttag 130741 ccagactgat ctcaatctcc tgacctcagg tgatctgccc accgcagcct cccaaagtgc 130801 tgggattaca agtgtgagcc accacgccag gcctctgtct tgataattac atttcaaagg 130861 aatggctccc aggtccttgg aaaagacatt cttggggtat aaaactggga agagtctggg 130921 aaaaggggca gagaaagaat ttataattcc aagtcttcta aagtaaatac tctaagaaaa 130981 gggaggttag gaatttatag ttgagaagtc tatctaaagt ttaataaagt ggaggagaac 131041 attaaggcca ttttagtcaa catacatgtt ctttttgtaa caatttcaac atttttcctt 131101 ttagcaaaca aaaaaatttt cttttggttg gaaccgctga tggcaagtta gcaatttttg 131161 aagataagac tgttaaggta aatgttgaat gcattctaca tctaaattta ttttaagtct 131221 tttgttttat atatatctca cacccctctt atgggattat aaactccctg agagcaagaa 131281 tcataaatta tgctgtattt gtattgcttc ataaaatctt gaacacagta gatcctctga 131341 aaatacttgc tgattgactg tatattttat atgaatgaac taagaataaa atgataaatg 131401 acatctgatt gataatattg ggaatggaaa taattcaatt tgtacataac tgaggcagat 131461 aattccttat aaatatattg tggaaaaaaa acaaaaatat acttaagttt taaatatggc 131521 ttgccattaa ctttttctta agcattgaag aaatcattta attttctttt cttcagattc 131581 ctatttagtc attaaagcat tcatttctct atccatctat tcatctttgg ttccatctat 131641 tcactcaact tcctacccgt tcattctcct attgccaaaa agcttattat ctgatgagag 131701 acagggaagt agagtataac ccttaggtta tttcttttgt aatttttaca tgggaaaaag 131761 aatagattga atgtaacaat aatatttcga atatgaccta aattttttta tgtataatat 131821 ttgtacatat ttatggggta catgtgatat gttgttacat gcatagaatg tgtaatgatc 131881 aagtcagggt atttaggata tccatcacca tgagcattta tttctctgtg ctgagaacat 131941 ttcaagtctc ctagttattt tgaaatgttt ttaactgtag tcactttatt gtactattga 132001 acattagaac ttattcctcc tatctaactg tatgtttgta cccgttaacc agcctccctt 132061 catcctcccc ttctcccaca cacccatatc ctcccaagcc tctggtaact atcattctac 132121 tctctacctc catgagatca acttttttag ctcccacata tgagtgagta catgtgatat 132181 ttgtctttct gtgcttggct tatttcactt aacataatga cctccagttc catccatgtt 132241 gctgtatatg acatgatttc attccttttt atggtcaaat agtattccgt tatgtaaata 132301 cacacatttt ctttatgcat tcattcattc atgggtgctt aggttgattc cacttttttt 132361 tttagctatt gtgaatagtg ctgcgataaa catggggata taggaatccc tttgatatac 132421 tgattccctt tcctttagat tagtatcagt agtaacattg ttggattgta tggtagttct 132481 atttttaatt tttttgagaa atcaccattt tgttttccgt agtggctata gcaatttaca 132541 tacacaccaa tagcatatgg gcattcgttt ttttccgcat ccttgccagc atgttatttt 132601 ttgtcttttt tataatagcc attctaattg ggtgaagaag atttcattgt ggttttcatt 132661 tgcattttta ctgatgatta gttaatgttg agcatttttt ttcatatatc cattggccat 132721 tactatgtct tcttttgcaa atgtctattt agatcctttg ccaacttttt gttttgtttt 132781 aagacagggt cttgctttct tacccaggct ggctcacagt ggcatgatca tagcttgttg 132841 cagccttgac cttctgcact caagtgatcc tccaacatca gcttcacgag tagctgggac 132901 tacaggcgtg tgctaccata cctggctgtt tattttttgt agagatgcgg ctccactatg 132961 ttgtccagac tgatctcaaa ctcctgggct caagcaatcc tcctgcctca tcttctcaaa 133021 gtgctgggat tacaggcatg agccaccata cccagccctt tgcctacttt taaatggagt 133081 tctttttttt ttttcctgtt gaattgcttt ttcgagtttc ttgtgtattc tggatgaata 133141 gtttgcaaat atttcctcac atttaatgga tcctctctat attgttgata gtttcctttg 133201 ctgtgcagaa gctttttagt ttattatagt cccatttgtc taattttgtt tttgttgcct 133261 gtgcttttgg gatcttaacc ataaactctt tgtctagacc aatgttctga aatgtttccc 133321 ctgtttcctt ttaatagttt catagcttct ggtcttacat ttaagtcttt aatccatctt 133381 gagttgaatt ttgtaaatgg tgagagagtg gggcctactt tcatccttct gcatactgat 133441 atccagcttt tccagcacaa tttattgaag gtggtattct ttctcccatg tatgcttttg 133501 gtgcctttgt tgaaaattag ttggctttaa atatgtgggt ttatttctgg gtcctctaca 133561 ttggtctacc tacctgtgtt tttgccaata ctgtgctgtt ttggttactc tagccttgta 133621 atatattttc aagtcaggta gtgtgatgcc tccagctttg ttctttttgc tcaggattgt 133681 tttggctatt ttggctcttt ttcggttcca ctcaagtttt agaaattttt ttttctattt 133741 ctgtgagaaa tatcattgga gctttcatgg gaatttcatt gaatctgtag attgctttgg 133801 gtagtatggt cattttaaca atattaattc ttccagtctg tgagcatgaa tatctttcca 133861 tttgtttgtg tcctcttcaa tttctttcat gtttttcagg tttccttata gagatcgttc 133921 atcatctttg ttaaatttac tcctaggtat tttattaatt ttttgtagct acttcaaatg 133981 ggattgcttt cttgattttt cagctacttt gttgttgttg ttgtatagat atgctactga 134041 tttctgtatg ttgattttga atcctttact atactcattt atcagaacta agagtttttt 134101 ttaatggaat ctttaggttt ttgagtttta attttaatat ctctaatcat ttaagatgga 134161 aagtagtttt ttgaaagcac agattttatg gagttttgtt ctgtggatac tcaatttgct 134221 gagtgtgttt tctttttttt ttggcaaagc ttaaaggagc tgctcctttg aagatactaa 134281 atataggaaa tgtcagtact ccattgatgt gtttgagtga atccacaaat tcaacggaaa 134341 gaaatgtaat gtggggagga tgtggcacaa agattttctc cttttctaat gatttcacca 134401 ttcagaaact cattgagaca agaacaagcc aactgtaagt tattttttat ctgtacaagt 134461 aatttatcat tatacttttg ttttttcctt ataatcatta ataatactgt tgataattca 134521 taaggaagat cttttaaaat gcataattta ttttctatca taaaattaaa ctttcattat 134581 aaaaaatttt gaaaattcca gaaagcagaa gggtattttt aagaagtcac tcaacctaat 134641 cacttttagg gataaaatat gtaaactcat tgtaatctta gtagtattta tcaatctaaa 134701 ttttttaaca atttttatta tctgtgtttc aaattagaca tgaaattgga agacaatcaa 134761 ctttgtattt caccaaattc acggactata catatgcaat ttgggtaact tccattaagt 134821 attgattgta ggaaagatag acagcaagta ttcttgcttg tccaaagttg tttctagatt 134881 tgataattat acagatgtct actcacagcc aagttagtga taccagtttc aaacaagaat 134941 aaaataaaat attaatataa acctctttca agtttgcttt ttttcagtgg tattttaatc 135001 aaatctttgc agttggttct tatttatcac attcctcagt gataagcagt ataggattgt 135061 ggataagagc ataaatcata gttcagatat tgctttgcca tctattggtt ttgtgaactt 135121 gggcaataac ctttcacatc ttcagtcatc tcttacctga ggattgtaat attctctatc 135181 tcaaagagat atttggagga gtaaataaga atgttaatat atggatctta attaacataa 135241 tgaccagcac ctggtaagct gtcaataaac attagctatt attattatta ttagctttga 135301 gtcacaaatc cctaccttag ggaatatcct ggtttcccat tatccatcaa atttcccaag 135361 attggcactt gggagtaatc tttgactcct ttgttttttg ctccctttat ccacttgacc 135421 ctcctaattg tcatttgaat ctttgtactt ctcactgttc tcagtgctgg taccatgggc 135481 cagactgcca tccattatct gtggccacat taacaagagc atccagttct caccctctga 135541 ttatactctc ttcaacatat tttcagcatt gcagacagag ggacctttct aaaatgtaca 135601 tcaatctgat gcaattttcc tgcttaaaac ccttcagtgg tatcccattg tcctttatat 135661 gaagtccaaa ttccttaaga cagcctactg ggcccttcat aattcagtgc ttgcttacct 135721 gtctagattc atattttgca agctttttgc catctgtgct ttacccaaac tgaaattgac 135781 tcagatctct aagacaacct gttattacct ttcaccccca acctttgaat gtggtcttct 135841 ccttacctgg atgactatta tgccccctgc ctatttcagt ccaagcacca cttgctctga 135901 gtggcaggtt aggtgacttt gtgcttccat tgcatctaat gttttccttc acagtagcta 135961 taattgtatt tgttaaagta gtttctcaat accactaaat ctactggctt tcaacattgg 136021 ctgtgcattt agaaaccact aggtagctga aaataatatg atacctgggc cctacctcag 136081 accaattaaa tcagaccagt taagcctggg atggggatca gatttttttt ttcaggttct 136141 caagtgattc taaagtatat ttgaggttaa gatatactgc ggagtgcagt gtattataag 136201 ttcccatgaa tgaggatttt tatttctgtc tttacatatt tatttactag tatgtggtta 136261 acatttggat caactcattc tcattctgta atacccacat tttaaaaaat gaatgtaaaa 136321 atgtctttta ttatttttat ttttcaaatt taattttaga ttccaggaat acatgtgcag 136381 gtttgttaca aaagtatagt gcgtgatgct gaggtttgga atacaactga actcacaacc 136441 cagaaagtgg gcatagtgct tgataggtag tttttcatct ttgctcccct tcctgtaatt 136501 tccactttta acccaattat acaagcctga aaacctttaa aaagaaaggg cctccagttt 136561 atttttttat ttcatagcac attttatggg attatggata tcagttaact ctttaaagtt 136621 ccatataaga tttggagcat agatgcttta ctagagagca tccataaaga tcaagctctc 136681 aaagatgctc atctcccaaa agagattggg acctagatgc ataaacactt aaatataaat 136741 atttgctctc ttctgaacaa gtatctcctg tggccttggt ctctacccca caaaacagac 136801 atcacatcac taatcagggg ttgcctcatc atcagtaccc tcatcatcat cagtacacac 136861 cgactgagag gcatgttggg taatgaaaga tgactgcctt tgaagccgga agactccata 136921 ggacttttgg agttctaggt ctgcaacatg tttttagtcc taggtcggca agcatgatgt 136981 tagtcatagg accttgagca aattatttag catttgtaca tgtttctttc tactattaaa 137041 aacattgaga tttatcatat tttatgtttt tttattaagg atcaagcaag ataacacaca 137101 aaagtatttt ataaaatacg gaaattccat gcaaaacttt gtcctaattg gaactatttt 137161 ctattaaata cagcaaatat ccaagaagga attacctaaa gcgtagtggc tctctgacaa 137221 cacatcatat ttttacctcc ttttccagaa tagaaagaaa agggggagga aaaaaaatct 137281 tcctactcta ggatatacta atgattgtta aatctttatg gtattttcat gttatctatc 137341 tgatttaaat gcaattttga ctatttttac atattcctcg ttgttcattc atactgtagt 137401 gccttcttct attcccccac ttaacagact gcttgattat atcagaggtg cttatctgtg 137461 caactgttta ctggacgagg ggtatgtaga aaatacattg tcctcatcct tatgaaatta 137521 cactcataat ctagtgtgag ggatgcacta ataaagacaa ttttatattt aataaggtct 137581 ataatatgac agtgacacca gtggggaaaa gggactggtt ggtctatttt gataggtcag 137641 ggaagaaatc cagaggagcc gacatttaag ttcatcctca aaggccaagt aggagtttgc 137701 catgctgatg tggcccaagg tagccaatct gtttgtgaaa tatgtacaat gccagatgtc 137761 ttaggttgaa agggaaatat tttaaggtgt tcgtaatttt tctttatgtt taaaagggga 137821 aaatggcaaa tattttactt tctgtttatg tttggatgat gtggattttt gttttctata 137881 atttgactgg cttaactgca aagatatccc ttgctttaaa atttgaagac actgcaacta 137941 aattttattt cagcatttta tattttataa ctctaggtat aaaaggctaa cacttaattt 138001 tctgagcatt catgaaacaa agttttgcaa gaacattcaa aagttacaga tataatattt 138061 ccttcagaaa tttagatata gtacaaaatt ctacaaagag ccacatagaa ttgaaactaa 138121 aagtaagacc aaagtaaaca ttggacataa tctttatttt attatcacaa gaaattaata 138181 taaagtaacc aaaagtaagt aaagtaccaa agcatgttat atattcaatt cagaatggtt 138241 agggaagaat atgaaataat tgcaatagtc tagcttgttt agttttcaaa atagtgtttt 138301 tacattaaga actaatataa ggttgtatta cacgtagaaa ttttaagaag aaaacaaata 138361 gtgatgactt tctatttttt tttctctgta ggttttctta tgcagctttc agtgattcca 138421 acatcataac agtggtggta gacactgctc tctatattgc taagcaaaat agccctgttg 138481 tggaagtgtg ggataagaaa actgaaaaac tctgtggact aatagactgc gtgcactttt 138541 taaggtaaat tctgtggttt ttaattttat tcccaaaaga attatctttg cacttcatgt 138601 gtcacagagg aaggattttt cttcctttct gcctctgaat agagaatttt tttaaaatgc 138661 agaaaaaaat ttgtaatgct tctcagcacc atcttttcag atcaagaaaa ttttgtcttc 138721 agaacataaa agaataggca cataatgtgc atagttttct catggtatta caaagaatgt 138781 tctcgaatga aaatactaca ttattgaaaa tgagcatatt ggagtctctg ctagctttga 138841 catagttctg tcacagtgtc aaatatacta tttataatta aattatgggc cccaggatta 138901 tctgctctaa agaaaaagag tcacaaaata atagacaaat atggggggaa atgcaatgga 138961 ctgaccgagg cgctaaggag tggggatcaa gaccccagaa tgagagcata gtgcttagtc 139021 tgatgcagcc tgtgagtgac aaatccatag caagcacatt ctttctgtgc tggtgctgag 139081 aaacaggacc attttcaagc ttatttgcta gccactttat atttttattt tgttttgatt 139141 ttaccatata gatctatgat actcttgaga acattttaga ttacacacta tatctgtaaa 139201 aggatacttc aaagtttcct gtcttagatt catctgacag tttttctatg gattgtgaga 139261 agggctcaca gtttatgttc agaagggcca acaggtctcc ttgataaagg gttccttact 139321 tcctgaagta ccaaaacgat taggattctt ttatttctgg acacttcatt tttgtcatga 139381 attagactat tcactggttc tgggaaaaaa ttcagtggtt tgtatcgata tcttttacat 139441 gtgaatgact ataattttat gttcctttgt aacattgaga cttcatgtaa aacttttgac 139501 tctaactttt ttttttcttt atcctgggca catgtatgct atttttactg aattagatag 139561 ctttggtatt tataaaaatt gtatccctct tattcataat ttcctgaaaa tgaagggcta 139621 ttgttatctt tgataattta tgcttcaagt aaagaagtgt ggctctttgg catctgtatt 139681 tagcaaaatt tgctttgtat aattttaatg atgcataatg gtggtggtgt catgttttaa 139741 taatttaaaa tgttgtttat gttatcatat gtaaatagca tttatctctt aattggtggt 139801 aaaattatta atgtatactt tatggttcta gggaggtaat ggtaaaagaa aacaaggaat 139861 caaaacacaa aatgtcttat tctgggagag tgaaaaccct ctgccttcag aagaacactg 139921 ctctttggat aggaactgga ggaggccata ttttactcct ggatctttca actcgtcgac 139981 ttatacgtgt aatttacaac ttttgtaatt cggtcagagt catgatgaca gcacagctag 140041 gcaagtttct ttcctttaga tatttttcat attctctaag tcttataaaa tatgccttta 140101 ttttacgttt acattttctc tgaactttcc agtgtcatat ggatggtctt ggagggtcac 140161 acagtgaaac ataagactgg tataaattgt gaatagggtc attacagaag tggagggagt 140221 aaatgctctc agtcccacaa gagaagcaga ttactgcagc tgaacactca gtttgggtct 140281 tacttgcttt tttccttttt acctaaggca aaaatgggaa atacatggta ttgaatatat 140341 tttacttttt gagcaaagaa aataaagaaa atgtttgttt taatcatagt ctagcctccc 140401 agcttgttaa agaatctcat ttggtttttc attctataac aaatcttttt tcttgcagca 140461 atacatgctg aactgcacaa cctacaaata ttgacaaatc atattttact caaactttgt 140521 ctttttttgc ttctattttt atatttaaat atgataaaat tgtgatagca cataaaatat 140581 attttctgca taaatatatt tgcgtcttcc tttgataata atttgtttta gaaaataaca 140641 ataatagcat atatacaaaa gtttacaaaa acgacactat ggggtttaat tctgaaaaaa 140701 actagaattt atgtaacttt agcaaataat gaatgttttg aacatggtga agaaaatata 140761 ttcattgcaa gtatatgtga aagaggaaca tgtgtttttc tagcaccttc acctattttt 140821 catttataga ctttagagtt gcacaggagt tacaattaga tgctcttaat gactgtaaac 140881 tattaagata catgtccaca caagcagagc agtaggtctc tcaataggtt gtctcagtag 140941 tcttattcta ccaaagttgt tgcattctct agttgaattg tatgtacttt gggacccaaa 141001 tagcttgctt ataactgaag ttatagtgga atgtctatgg gttatagttt gattttaaaa 141061 taaagatcaa ttggaggata gcctacaagg tgctgcatga gctggcttca ctgtacctct 141121 cctgcctcca tcatctacca cattcctacc agatcttgct tgtctagatg aacatccagt 141181 tcttctcaat tactatgcta tattttgcct cagggatttg cacatgctgt ttatttcttt 141241 gcttagttaa catagctttt tttcatgctt atttaactca tatgctttga aatgttagct 141301 cctgtgtcaa aacctctgag aagccaacac tgattaggtc aaagttcccg ctttgggttc 141361 ccataacagc tttcttgcat agttttgatc atggtcatat ttttttttca ttaatgctag 141421 tctcttcact agaatataaa ctccaagacg gttgggttag tgtgtttttg tttacccctt 141481 ttttcccagg atctagccta gggcctacat agaagacttt tgatgcaaat ttgttgaata 141541 aattagtgaa tgatttgaaa agaaaatatg atattttgac atagtatcag tatatccatc 141601 catctaagtg tccatctaaa tactcatatt tgtactaaat actcatattt gtaccttacc 141661 atatccaaag aacttttcac acacattacc tcgtttaaca ttgtaacttt gggaagggta 141721 atgtataaat actgagccta ttttatagaa aggttaagct gttttctcag actcacataa 141781 ttaagaattg cagcaaggag tggatcacag attttgttat tttttaaaaa aatgctggtc 141841 tttattcaat ataattgaag ggtcacctag aaaatagaat tgtgaattca gttccaaggt 141901 atttgtgtct taaactatga acaactttac ttttttttca ggccagttta atatatagtt 141961 ttaacagaaa acttacatat tttgtttttg taaaggaagc cttaaaaatg tcatgctggt 142021 attgggctac aaccggaaaa atactgaagg tacacaaaag cagaaaggta acatttagaa 142081 ggatactgtt ttccaaacag ggcaatgatg tgaatgatgg taacatatta tgtgtttcat 142141 aaatttgtag aaaatattac atatggtata atcaggaatt ttaattggta gtttatagtg 142201 taaagaactt agacataaat tttcaaaatt acaagtgata tgaagtgtta aatatttata 142261 ttttcagctg aagtagaggt gtcaatcact agctcaacct taaacgaaat gtgaatattt 142321 tttacaactt atctatatct acataatgtc taattttgaa cagtgtttga aaaagctttt 142381 atttctttta gaatatgaaa tgttaattta ttaaatgttg atactctatt tgaaatttaa 142441 tagtttctat aatgtattat aaaacttttc caagtatagt tttttataaa taataattta 142501 gtacattagt tatagctgtg tttatattta catttatcta agtcaactaa aaatacatga 142561 gccaaactga aataaaataa gaatgtttta tgatggatct ttgaaacatg atttcatttt 142621 tttctttttc tagagataca atcttgcttg accgtttggg acatcaatct tccacatgaa 142681 gtgcaaaatt tagaaaaaca cattgaagtg agaaaagaat tagctgaaaa aatgagacga 142741 acatctgttg agtaagagag aaataggaat tgtctttgga taggaaaatt attctctcct 142801 cttgtaaata tttattttaa aaatgttcac atggaaaggg tactcacatt ttttgaaata 142861 gctcgtgtgt atgaaggaat gttattattt ttaatttaaa tatatgtaaa aatacttacc 142921 agtaaatgtg tattttaaag aactatttaa aacacaatgt tatatttctt ataaatacca 142981 gttactttcg ttcattaatt aatgaaaata aatctgtgaa gtacctaatt taagtactca 143041 tactaaaatt tataaggccg ataatttttt gttttcttgt ctgtaatgga ggtaaacttt 143101 attttaaatt ctgtgcttaa gacaggacta ttgcttgtcg atttttctag aaatctgcac 143161 ggtataatga aaatattaag acagtttccc atgtaatgta ttccttctta gattgcatcg 143221 aaatgcacta tcatatatgc ttgtaaatat tcaaatgaat ttgcactaat aaagtccttt 143281 gttggtatgt gaattctctt tgttgctgtt gcaaacagtg catcttacac aacttcactc 143341 aattcaaaag aaaactccat taaaagtact aatgaaaaaa catgacatac tgtcaaagtc 143401 ctcatatcta ggaaagacac agaaactctc tttgtcacag aaactctctg tgtctttcct 143461 agacataata gagttgtttt tcaactctat gtttgaatgt ggataccctg aattttgtat 143521 aattagtgta aatacagtgt tcagtccttc aagtgatatt tttatttttt tattcatacc 143581 actagctact tgttttctaa tctgcttcat tctaatgctt atattcatct tttccctaaa 143641 tttgtgatgc tgcagatcct acatcattca gatagaaacc tttttttttt tcagaattat 143701 agaattccac agctcctacc aagaccatga ggataaatat ctaacacttt tcagttgctg 143761 aaggagaaag gagctttagt tatgatggat aaaaatatct gccaccctag gcttccaaat 143821 tatacttaaa ttgtttacat agcttaccac aataggagta tcagggccaa atacctatgt 143881 aataatttga ggtcatttct gctttaggaa aagtactttc ggtaaattct ttggccctga 143941 ccagtattca ttatttcaga taattccctg tgataggaca actagtacat ttaatattct 144001 cagaacttat ggcattttac tatgtgaaaa ctttaaattt atttatatta agggtaatca 144061 aattcttaaa gatgaaagat tttctgtatt ttaaaggaag ctatgcttta acttgttatg 144121 taattaacaa aaaaatcata tataatagag ctctttgttc cagtgttatc tctttcattg 144181 ttactttgta tttgcaattt tttttaccaa agacaaatta aaaaaatgaa taccatattt 144241 aaatggaata ataaaggttt tttaaaaact ttaaa

For example, the nucleotide sequence corresponding to the mRNA of the human LRRK2 is depicted in SEQ ID NO: 10 (9239 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Sequence information related to LRRK2 is accessible in public databases by GenBank Accession number NM_198578.3 (nucleotide).

SEQ ID NO: 10:    1 gcgctggctg cgggcggtga gctgagctcg cccccgggga gctgtggccg gcgcccctgc   61 cggttccctg agcagcggac gttcatgctg ggagggcggc gggttggaag caggtgccac  121 c atg gctagt ggcagctgtc aggggtgcga agaggacgag gaaactctga agaagttgat  181 agtcaggctg aacaatgtcc aggaaggaaa acagatagaa acgctggtcc aaatcctgga  241 ggatctgctg gtgttcacgt actccgagca cgcctccaag ttatttcaag gcaaaaatat  301 ccatgtgcct ctgttgatcg tcttggactc ctatatgaga gtcgcgagtg tgcagcaggt  361 gggttggtca cttctgtgca aattaataga agtctgtcca ggtacaatgc aaagcttaat  421 gggaccccag gatgttggaa atgattggga agtccttggt gttcaccaat tgattcttaa  481 aatgctaaca gttcataatg ccagtgtaaa cttgtcagtg attggactga agaccttaga  541 tctcctccta acttcaggta aaatcacctt gctgatattg gatgaagaaa gtgatatttt  601 catgttaatt tttgatgcca tgcactcatt tccagccaat gatgaagtcc agaaacttgg  661 atgcaaagct ttacatgtgc tgtttgagag agtctcagag gagcaactga ctgaatttgt  721 tgagaacaaa gattatatga tattgttaag tgcgttaaca aattttaaag atgaagagga  781 aattgtgctt catgtgctgc attgtttaca ttccctagcg attccttgca ataatgtgga  841 agtcctcatg agtggcaatg tcaggtgtta taatattgtg gtggaagcta tgaaagcatt  901 ccctatgagt gaaagaattc aagaagtgag ttgctgtttg ctccataggc ttacattagg  961 taattttttc aatatcctgg tattaaacga agtccatgag tttgtggtga aagctgtgca 1021 gcagtaccca gagaatgcag cattgcagat ctcagcgctc agctgtttgg ccctcctcac 1081 tgagactatt ttcttaaatc aagatttaga ggaaaagaat gagaatcaag agaatgatga 1141 tgagggggaa gaagataaat tgttttggct ggaagcctgt tacaaagcat taacgtggca 1201 tagaaagaac aagcacgtgc aggaggccgc atgctgggca ctaaataatc tccttatgta 1261 ccaaaacagt ttacatgaga agattggaga tgaagatggc catttcccag ctcataggga 1321 agtgatgctc tccatgctga tgcattcttc atcaaaggaa gttttccagg catctgcgaa 1381 tgcattgtca actctcttag aacaaaatgt taatttcaga aaaatactgt tatcaaaagg 1441 aatacacctg aatgttttgg agttaatgca gaagcatata cattctcctg aagtggctga 1501 aagtggctgt aaaatgctaa atcatctttt tgaaggaagc aacacttccc tggatataat 1561 ggcagcagtg gtccccaaaa tactaacagt tatgaaacgt catgagacat cattaccagt 1621 gcagctggag gcgcttcgag ctattttaca ttttatagtg cctggcatgc cagaagaatc 1681 cagggaggat acagaatttc atcataagct aaatatggtt aaaaaacagt gtttcaagaa 1741 tgatattcac aaactggtcc tagcagcttt gaacaggttc attggaaatc ctgggattca 1801 gaaatgtgga ttaaaagtaa tttcttctat tgtacatttt cctgatgcat tagagatgtt 1861 atccctggaa ggtgctatgg attcagtgct tcacacactg cagatgtatc cagatgacca 1921 agaaattcag tgtctgggtt taagtcttat aggatacttg attacaaaga agaatgtgtt 1981 cataggaact ggacatctgc tggcaaaaat tctggtttcc agcttatacc gatttaagga 2041 tgttgctgaa atacagacta aaggatttca gacaatctta gcaatcctca aattgtcagc 2101 atctttttct aagctgctgg tgcatcattc atttgactta gtaatattcc atcaaatgtc 2161 ttccaatatc atggaacaaa aggatcaaca gtttctaaac ctctgttgca agtgttttgc 2221 aaaagtagct atggatgatt acttaaaaaa tgtgatgcta gagagagcgt gtgatcagaa 2281 taacagcatc atggttgaat gcttgcttct attgggagca gatgccaatc aagcaaagga 2341 gggatcttct ttaatttgtc aggtatgtga gaaagagagc agtcccaaat tggtggaact 2401 cttactgaat agtggatctc gtgaacaaga tgtacgaaaa gcgttgacga taagcattgg 2461 gaaaggtgac agccagatca tcagcttgct cttaaggagg ctggccctgg atgtggccaa 2521 caatagcatt tgccttggag gattttgtat aggaaaagtt gaaccttctt ggcttggtcc 2581 tttatttcca gataagactt ctaatttaag gaaacaaaca aatatagcat ctacactagc 2641 aagaatggtg atcagatatc agatgaaaag tgctgtggaa gaaggaacag cctcaggcag 2701 cgatggaaat ttttctgaag atgtgctgtc taaatttgat gaatggacct ttattcctga 2761 ctcttctatg gacagtgtgt ttgctcaaag tgatgacctg gatagtgaag gaagtgaagg 2821 ctcatttctt gtgaaaaaga aatctaattc aattagtgta ggagaatttt accgagatgc 2881 cgtattacag cgttgctcac caaatttgca aagacattcc aattccttgg ggcccatttt 2941 tgatcatgaa gatttactga agcgaaaaag aaaaatatta tcttcagatg attcactcag 3001 gtcatcaaaa cttcaatccc atatgaggca ttcagacagc atttcttctc tggcttctga 3061 gagagaatat attacatcac tagacctttc agcaaatgaa ctaagagata ttgatgccct 3121 aagccagaaa tgctgtataa gtgttcattt ggagcatctt gaaaagctgg agcttcacca 3181 gaatgcactc acgagctttc cacaacagct atgtgaaact ctgaagagtt tgacacattt 3241 ggacttgcac agtaataaat ttacatcatt tccttcttat ttgttgaaaa tgagttgtat 3301 tgctaatctt gatgtctctc gaaatgacat tggaccctca gtggttttag atcctacagt 3361 gaaatgtcca actctgaaac agtttaacct gtcatataac cagctgtctt ttgtacctga 3421 gaacctcact gatgtggtag agaaactgga gcagctcatt ttagaaggaa ataaaatatc 3481 agggatatgc tcccccttga gactgaagga actgaagatt ttaaacctta gtaagaacca 3541 catttcatcc ctatcagaga actttcttga ggcttgtcct aaagtggaga gtttcagtgc 3601 cagaatgaat tttcttgctg ctatgccttt cttgcctcct tctatgacaa tcctaaaatt 3661 atctcagaac aaattttcct gtattccaga agcaatttta aatcttccac acttgcggtc 3721 tttagatatg agcagcaatg atattcagta cctaccaggt cccgcacact ggaaatcttt 3781 gaacttaagg gaactcttat ttagccataa tcagatcagc atcttggact tgagtgaaaa 3841 agcatattta tggtctagag tagagaaact gcatctttct cacaataaac tgaaagagat 3901 tcctcctgag attggctgtc ttgaaaatct gacatctctg gatgtcagtt acaacttgga 3961 actaagatcc tttcccaatg aaatggggaa attaagcaaa atatgggatc ttcctttgga 4021 tgaactgcat cttaactttg attttaaaca tataggatgt aaagccaaag acatcataag 4081 gtttcttcaa cagcgattaa aaaaggctgt gccttataac cgaatgaaac ttatgattgt 4141 gggaaatact gggagtggta aaaccacctt attgcagcaa ttaatgaaaa ccaagaaatc 4201 agatcttgga atgcaaagtg ccacagttgg catagatgtg aaagactggc ctatccaaat 4261 aagagacaaa agaaagagag atctcgtcct aaatgtgtgg gattttgcag gtcgtgagga 4321 attctatagt actcatcccc attttatgac gcagcgagca ttgtaccttg ctgtctatga 4381 cctcagcaag ggacaggctg aagttgatgc catgaagcct tggctcttca atataaaggc 4441 tcgcgcttct tcttcccctg tgattctcgt tggcacacat ttggatgttt ctgatgagaa 4501 gcaacgcaaa gcctgcatga gtaaaatcac caaggaactc ctgaataagc gagggttccc 4561 tgccatacga gattaccact ttgtgaatgc caccgaggaa tctgatgctt tggcaaaact 4621 tcggaaaacc atcataaacg agagccttaa tttcaagatc cgagatcagc ttgttgttgg 4681 acagctgatt ccagactgct atgtagaact tgaaaaaatc attttatcgg agcgtaaaaa 4741 tgtgccaatt gaatttcccg taattgaccg gaaacgatta ttacaactag tgagagaaaa 4801 tcagctgcag ttagatgaaa atgagcttcc tcacgcagtt cactttctaa atgaatcagg 4861 agtccttctt cattttcaag acccagcact gcagttaagt gacttgtact ttgtggaacc 4921 caagtggctt tgtaaaatca tggcacagat tttgacagtg aaagtggaag gttgtccaaa 4981 acaccctaag ggcattattt cgcgtagaga tgtggaaaaa tttctttcaa aaaaaaggaa 5041 atttccaaag aactacatgt cacagtattt taagctccta gaaaaattcc agattgcttt 5101 gccaatagga gaagaatatt tgctggttcc aagcagtttg tctgaccaca ggcctgtgat 5161 agagcttccc cattgtgaga actctgaaat tatcatccga ctatatgaaa tgccttattt 5221 tccaatggga ttttggtcaa gattaatcaa tcgattactt gagatttcac cttacatgct 5281 ttcagggaga gaacgagcac ttcgcccaaa cagaatgtat tggcgacaag gcatttactt 5341 aaattggtct cctgaagctt attgtctggt aggatctgaa gtcttagaca atcatccaga 5401 gagtttctta aaaattacag ttccttcttg tagaaaaggc tgtattcttt tgggccaagt 5461 tgtggaccac attgattctc tcatggaaga atggtttcct gggttgctgg agattgatat 5521 ttgtggtgaa ggagaaactc tgttgaagaa atgggcatta tatagtttta atgatggtga 5581 agaacatcaa aaaatcttac ttgatgactt gatgaagaaa gcagaggaag gagatctctt 5641 agtaaatcca gatcaaccaa ggctcaccat tccaatatct cagattgccc ctgacttgat 5701 tttggctgac ctgcctagaa atattatgtt gaataatgat gagttggaat ttgaacaagc 5761 tccagagttt ctcctaggtg atggcagttt tggatcagtt taccgagcag cctatgaagg 5821 agaagaagtg gctgtgaaga tttttaataa acatacatca ctcaggctgt taagacaaga 5881 gcttgtggtg ctttgccacc tccaccaccc cagtttgata tctttgctgg cagctgggat 5941 tcgtccccgg atgttggtga tggagttagc ctccaagggt tccttggatc gcctgcttca 6001 gcaggacaaa gccagcctca ctagaaccct acagcacagg attgcactcc acgtagctga 6061 tggtttgaga tacctccact cagccatgat tatataccga gacctgaaac cccacaatgt 6121 gctgcttttc acactgtatc ccaatgctgc catcattgca aagattgctg actacggcat 6181 tgctcagtac tgctgtagaa tggggataaa aacatcagag ggcacaccag ggtttcgtgc 6241 acctgaagtt gccagaggaa atgtcattta taaccaacag gctgatgttt attcatttgg 6301 tttactactc tatgacattt tgacaactgg aggtagaata gtagagggtt tgaagtttcc 6361 aaatgagttt gatgaattag aaatacaagg aaaattacct gatccagtta aagaatatgg 6421 ttgtgcccca tggcctatgg ttgagaaatt aattaaacag tgtttgaaag aaaatcctca 6481 agaaaggcct acttctgccc aggtctttga cattttgaat tcagctgaat tagtctgtct 6541 gacgagacgc attttattac ctaaaaacgt aattgttgaa tgcatggttg ctacacatca 6601 caacagcagg aatgcaagca tttggctggg ctgtgggcac accgacagag gacagctctc 6661 atttcttgac ttaaatactg aaggatacac ttctgaggaa gttgctgata gtagaatatt 6721 gtgcttagcc ttggtgcatc ttcctgttga aaaggaaagc tggattgtgt ctgggacaca 6781 gtctggtact ctcctggtca tcaataccga agatgggaaa aagagacata ccctagaaaa 6841 gatgactgat tctgtcactt gtttgtattg caattccttt tccaagcaaa gcaaacaaaa 6901 aaattttctt ttggttggaa ccgctgatgg caagttagca atttttgaag ataagactgt 6961 taagcttaaa ggagctgctc ctttgaagat actaaatata ggaaatgtca gtactccatt 7021 gatgtgtttg agtgaatcca caaattcaac ggaaagaaat gtaatgtggg gaggatgtgg 7081 cacaaagatt ttctcctttt ctaatgattt caccattcag aaactcattg agacaagaac 7141 aagccaactg ttttcttatg cagctttcag tgattccaac atcataacag tggtggtaga 7201 cactgctctc tatattgcta agcaaaatag ccctgttgtg gaagtgtggg ataagaaaac 7261 tgaaaaactc tgtggactaa tagactgcgt gcacttttta agggaggtaa tggtaaaaga 7321 aaacaaggaa tcaaaacaca aaatgtctta ttctgggaga gtgaaaaccc tctgccttca 7381 gaagaacact gctctttgga taggaactgg aggaggccat attttactcc tggatctttc 7441 aactcgtcga cttatacgtg taatttacaa cttttgtaat tcggtcagag tcatgatgac 7501 agcacagcta ggaagcctta aaaatgtcat gctggtattg ggctacaacc ggaaaaatac 7561 tgaaggtaca caaaagcaga aagagataca atcttgcttg accgtttggg acatcaatct 7621 tccacatgaa gtgcaaaatt tagaaaaaca cattgaagtg agaaaagaat tagctgaaaa 7681 aatgagacga acatctgttg agtaagagag aaataggaat tgtctttgga taggaaaatt 7741 attctctcct cttgtaaata tttattttaa aaatgttcac atggaaaggg tactcacatt 7801 ttttgaaata gctcgtgtgt atgaaggaat gttattattt ttaatttaaa tatatgtaaa 7861 aatacttacc agtaaatgtg tattttaaag aactatttaa aacacaatgt tatatttctt 7921 ataaatacca gttactttcg ttcattaatt aatgaaaata aatctgtgaa gtacctaatt 7981 taagtactca tactaaaatt tataaggccg ataatttttt gttttcttgt ctgtaatgga 8041 ggtaaacttt attttaaatt ctgtgcttaa gacaggacta ttgcttgtcg atttttctag 8101 aaatctgcac ggtataatga aaatattaag acagtttccc atgtaatgta ttccttctta 8161 gattgcatcg aaatgcacta tcatatatgc ttgtaaatat tcaaatgaat ttgcactaat 8221 aaagtccttt gttggtatgt gaattctctt tgttgctgtt gcaaacagtg catcttacac 8281 aacttcactc aattcaaaag aaaactccat taaaagtact aatgaaaaaa catgacatac 8341 tgtcaaagtc ctcatatcta ggaaagacac agaaactctc tttgtcacag aaactctctg 8401 tgtctttcct agacataata gagttgtttt tcaactctat gtttgaatgt ggataccctg 8461 aattttgtat aattagtgta aatacagtgt tcagtccttc aagtgatatt tttatttttt 8521 tattcatacc actagctact tgttttctaa tctgcttcat tctaatgctt atattcatct 8581 tttccctaaa tttgtgatgc tgcagatcct acatcattca gatagaaacc tttttttttt 8641 tcagaattat agaattccac agctcctacc aagaccatga ggataaatat ctaacacttt 8701 tcagttgctg aaggagaaag gagctttagt tatgatggat aaaaatatct gccaccctag 8761 gcttccaaat tatacttaaa ttgtttacat agcttaccac aataggagta tcagggccaa 8821 atacctatgt aataatttga ggtcatttct gctttaggaa aagtactttc ggtaaattct 8881 ttggccctga ccagtattca ttatttcaga taattccctg tgataggaca actagtacat 8941 ttaatattct cagaacttat ggcattttac tatgtgaaaa ctttaaattt atttatatta 9001 agggtaatca aattcttaaa gatgaaagat tttctgtatt ttaaaggaag ctatgcttta 9061 acttgttatg taattaacaa aaaaatcata tataatagag ctctttgttc cagtgttatc 9121 tctttcattg ttactttgta tttgcaattt tttttaccaa agacaaatta aaaaaatgaa 9181 taccatattt aaatggaata ataaaggttt tttaaaaact ttaaaaaaaa aaaaaaaaa

For example, the polypeptide sequence corresponding to human LRRK2 is encoded by the nucleic acid sequence of SEQ ID NO: 10 and is depicted in SEQ ID NO: 11 (2527aa). Sequence information related to LRRK2 is accessible in public databases by GenBank Accession numbers NP_940980.3 (protein).

   1 MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSEH ASKLFQGKNI   61 HVPLLIVLDS YMRVASVQQV GWSLLCKLIE VCPGTMQSLM GPQDVGNDWE VLGVHQLILK  121 MLTVHNASVN LSVIGLKTLD LLLTSGKITL LILDEESDIF MLIFDAMHSF PANDEVQKLG  181 CKALHVLFER VSEEQLTEFV ENKDYMILLS ALTNFKDEEE IVLHVLHCLH SLAIPCNNVE  241 VLMSGNVRCY NIVVEAMKAF PMSERIQEVS CCLLHRLTLG NFFNILVLNE VHEFVVKAVQ  301 QYPENAALQI SALSCLALLT ETIFLNQDLE EKNENQENDD EGEEDKLFWL EACYKALTWH  361 RKNKHVQEAA CWALNNLLMY QNSLHEKIGD EDGHFPAHRE VMLSMLMHSS SKEVFQASAN  421 ALSTLLEQNV NFRKILLSKG IHLNVLELMQ KHIHSPEVAE SGCKMLNHLF EGSNTSLDIM  481 AAVVPKILTV MKRHETSLPV QLEALRAILH FIVPGMPEES REDTEFHHKL NMVKKQCFKN  541 DIHKLVLAAL NRFIGNPGIQ KCGLKVISSI VHFPDALEML SLEGAMDSVL HTLQMYPDDQ  601 EIQCLGLSLI GYLITKKNVF IGTGHLLAKI LVSSLYRFKD VAEIQTKGFQ TILAILKLSA  661 SFSKLLVHHS FDLVIFHQMS SNIMEQKDQQ FLNLCCKCFA KVAMDDYLKN VMLERACDQN  721 NSIMVECLLL LGADANQAKE GSSLICQVCE KESSPKLVEL LLNSGSREQD VRKALTISIG  781 KGDSQIISLL LRRLALDVAN NSICLGGFCI GKVEPSWLGP LFPDKTSNLR KQTNIASTLA  841 RMVIRYQMKS AVEEGTASGS DGNFSEDVLS KFDEWTFIPD SSMDSVFAQS DDLDSEGSEG  901 SFLVKKKSNS ISVGEFYRDA VLQRCSPNLQ RHSNSLGPIF DHEDLLKRKR KILSSDDSLR  961 SSKLQSHMRH SDSISSLASE REYITSLDLS ANELRDIDAL SQKCCISVHL EHLEKLELHQ 1021 NALTSFPQQL CETLKSLTHL DLHSNKFTSF PSYLLKMSCI ANLDVSRNDI GPSVVLDPTV 1081 KCPTLKQFNL SYNQLSFVPE NLTDVVEKLE QLILEGNKIS GICSPLRLKE LKILNLSKNH 1141 ISSLSENFLE ACPKVESFSA RMNFLAAMPF LPPSMTILKL SQNKFSCIPE AILNLPHLRS 1201 LDMSSNDIQY LPGPAHWKSL NLRELLFSHN QISILDLSEK AYLWSRVEKL HLSHNKLKEI 1261 PPEIGCLENL TSLDVSYNLE LRSFPNEMGK LSKIWDLPLD ELHLNFDFKH IGCKAKDIIR 1321 FLQQRLKKAV PYNRMKLMIV GNTGSGKTTL LQQLMKTKKS DLGMQSATVG IDVKDWPIQI 1381 RDKRKRDLVL NVWDFAGREE FYSTHPHFMT QRALYLAVYD LSKGQAEVDA MKPWLFNIKA 1441 RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA TEESDALAKL 1501 RKTIINESLN FKIRDQLVVG QLIPDCYVEL EKIILSERKN VPIEFPVIDR KRLLQLVREN 1561 QLQLDENELP HAVHFLNESG VLLHFQDPAL QLSDLYFVEP KWLCKIMAQI LTVKVEGCPK 1621 HPKGIISRRD VEKFLSKKRK FPKNYMSQYF KLLEKFQIAL PIGEEYLLVP SSLSDHRPVI 1681 ELPHCENSEI IIRLYEMPYF PMGFWSRLIN RLLEISPYML SGRERALRPN RMYWRQGIYL 1741 NWSPEAYCLV GSEVLDNHPE SFLKITVPSC RKGCILLGQV VDHIDSLMEE WFPGLLEIDI 1801 CGEGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI 1861 LADLPRNIML NNDELEFEQA PEFLLGDGSF GSVYRAAYEG EEVAVKIFNK HTSLRLLRQE 1921 LVVLCHLHHP SLISLLAAGI RPRMLVMELA SKGSLDRLLQ QDKASLTRTL QHRIALHVAD 1981 GLRYLHSAMI IYRDLKPHNV LLFTLYPNAA IIAKIADYGI AQYCCRMGIK TSEGTPGFRA 2041 PEVARGNVIY NQQADVYSFG LLLYDILTTG GRIVEGLKFP NEFDELEIQG KLPDPVKEYG 2101 CAPWPMVEKL IKQCLKENPQ ERPTSAQVFD ILNSAELVCL TRRILLPKNV IVECMVATHH 2161 NSRNASIWLG CGHTDRGQLS FLDLNTEGYT SEEVADSRIL CLALVHLPVE KESWIVSGTQ 2221 SGTLLVINTE DGKKRHTLEK MTDSVTCLYC NSFSKQSKQK NFLLVGTADG KLAIFEDKTV 2281 KLKGAAPLKI LNIGNVSTPL MCLSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT 2341 SQLFSYAAFS DSNIITVVVD TALYIAKQNS PVVEVWDKKT EKLCGLIDCV HFLREVMVKE 2401 NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCNSVRVMMT 2461 AQLGSLKNVM LVLGYNRKNT EGTQKQKEIQ SCLTVWDINL PHEVQNLEKH IEVRKELAEK 2521 MRRTSVE

The invention provides for a nucleic acid encoding a VPS35 protein, or fragment thereof.

For example, the human genomic nucleotide sequence corresponding to the sense strand of the human VPS35 gene is depicted in SEQ ID NO: 12 (29556 bp). Sequence information related to VPS35 is accessible in public databases by GenBank Accession number NG_029970.1 (nucleotide).

SEQ ID NO: 12:     1 gctagagagg gcggggcttg gaggggccgc agcgtcacat gaccgcggga ggctacgcgc    61 ggggcgggtg ctgcttgctg caggctctgg ggagtcgcca tggtgagtgc tgagggggca   121 gtggcacctg ggtcgaccct ccttgtagcc cctgctctct cccaccgccc cgcactccag   181 cgagtggaga aggggcccca cagaccgttc gggattaaga ccagcccgat ttggcctgcg   241 ggatagggga cagcaggagg aaggccgcgg gcaggctgat ccgggccggg gtgggcggcg   301 gctcttggct gcggccgttg ctgagagacg gggcggcctc tctgtggggt tgacttggca   361 tgtaggcttt ggggtccatg aaggcctgcg gcctccttta agtggaatcg gtcacctgcc   421 taccacgagg ggaccggtag tcctaggtct gagcgtctgg cccccggggc gcgtggaggc   481 cctgagactc ggaggtggcg ccgggacccg cccagatgtt gcgtttctac ctttgtgcct   541 agttgtgctc ggccgtcccc acgccctcct ggaggggtcg cagtgattcc ttggcctttc   601 ttggcctcat acccgccttc ggctgcagtg tttgtcagcg agttctgggg acctgcttac   661 atgaatttcc tggaaggact caggctgtct tctaatcctg acggtcgcaa aggagactga   721 ttgtttactt tagcatttgt gcattgggcg caccttgcct cttttgtctc gccattgata   781 aaatccaagt atttgacttg ctggaagcag tacttctcct tagggcccgt ctatgacggc   841 agcaaatcgt ggtgtggctg ttggccggta aacttgaact tcctcaaaat gtgaatcttt   901 gtgtctggtt cccacaaagg caagttgtca cttgcatttt attagcgttt aacatagcct   961 gcactgtgta aataaatttt ttgagtatat actgtatgtc cgctttaatt accttactca  1021 ctctgtgtag ataggcttct gtaaatctgt aagcctggaa acagatttca ctttaaatgt  1081 cttaatgcca gaaaggatta agtgttttac aaatactatt ttcatataac gtgttgccgt  1141 acaaggtgat tttgcctgtt tctcaggatt tttataattg ggaattgata caagaccggc  1201 gcaaaattta actttaggat ttgtgtgttt ccagcgttta tggattgaca tttatattgt  1261 tttgtaatgg aaaacactta attgaggatg tattacacac tccgattctt tgttgggtga  1321 accagttggg agcaatcagc cagacacaca gtcttgtcct catgaatttt attgggaaag  1381 gaaacttgag tattcgttct ttcacagtca ctgtctataa tttaggcagc tcgcttagtc  1441 ttcttggtgt cgcacttttg catccgaaag caagtgttgg cggccccttt ggcccttccc  1501 agccctaaaa ttccagaatc cacttcaatt catcaaacat cttgtgtgcc ttcagtgtca  1561 cggactgtgg ccctgggtac caatgtcgaa cttaagaggc tactggggga gacacgcttg  1621 tagaacttaa ctgtaataca gtgtgtggat tgctttacta acattgtcaa ctcagtgctt  1681 tggggtcatg gagggatagt tttgcctggg gattttggga aggactacag gaagtaacgt  1741 gaactgagac tataaagagg gtggaaagaa attctccagg tggaaagaaa aggtattgct  1801 aatagatgaa gccaaaacgt tcagtgtgct ccatcagagg attcttgggg atatgtattg  1861 gaaatgaagt tagagccgtt tggtgttcaa attatatttt gaagttgtca tgccctcatc  1921 atccttattt ctctctactt cagttgtcaa tgtatgccat gtcatcattt ttaaaatagt  1981 tctttcatta acgtgatatt ctgtaccttt gtttgaagaa atgagcgtta catttggttt  2041 acattgtact gtcagattac atctacagca tataagcagg gaggttgaac cacaataggt  2101 ttctaacata agtggagata attatttgta tctaaactga ataataatta tagcacttat  2161 taagtgctta ttgctaagta gtaggcacgt tttgagcttt ggtttcattt gttcttcata  2221 acagtggtgt actctcatct ctgtttcata gaagaggaaa gaaaagcaca ttgtttcagt  2281 ggagagtagt tttccaagtg atcagttcat gatctttgct ttgctctgca cattagaact  2341 gtagacttga gagggctgtt tgtgggtctc aagctaaaat gggactaatc acaaatgtta  2401 atttgattcc cattctgtag ggtttccatt ttcttctgat tcatgtagct gtgaagtacg  2461 gtcatttaca aatggaaaac cactttcatg attgaggaaa cattggttaa ttggcttgtg  2521 tttaatgata gcgtgctcat attataactg ttaaggctct atgtgacact tcatagtaag  2581 gcatcaagaa tagcccttat aatagctgtt gctagcataa ctacactgtt ttatgagtaa  2641 tatataaaaa tagattgctt tactatagct ttatgtcttc acttgtagct tactttgtaa  2701 taagtcatat tctgtaatct tcaacatttt gtatttacat ttctatttta agctgagttg  2761 agacaaaata gaagaatttt cctaaaattg cattcttttg tttacacatc tctaaatctt  2821 tctagtcatt ctatacaaat gtttttggaa ctgattgtat cctctttgta gccacagttt  2881 catttgctat ataataaaaa ttatcataca caacagccaa aaggtggaaa caacccatat  2941 gtccatggat aaacaaaatg tagtgtgtgt gtatatatat agatatataa tgaaatactt  3001 cgtttttaaa aggagggaaa ttctggcaca tactgcaaca tggatgaaca ttgaagatat  3061 tatggtcagt gaaataagcg agacataaaa gtaaaaatat tgtatgattt cacttaaatg  3121 aagtatatag agtagtcaaa ctcagagaaa agaaggtaga attgcctggg ggttcggaat  3181 gggagtttaa tggctacagt ttctatttgc atttgtaaag ataaaaagtt ttagagatag  3241 atggtggtaa ttgtattaat gtgaatgtac ctaatgccac tgaactgtac atttaaatgt  3301 taaaatggta aattttaggt acattttacc acaacaagaa gtcattttta attaaaaata  3361 tccagatgta tcataaagaa aaataaaaaa aattcagatg tatcactgtt tatctctaaa  3421 tggatcaatt gaacttaatg aaatccattg attcaaatta ttattaaatc tattgcgtcc  3481 agaggtaagg agccaaaaaa ttccaaatga tggcctggtt tcactaaagt tcagagaaga  3541 ctagcccatg atgaatagta aatttcatta agtcagagtc tttaaatgct ggtgtcatcc  3601 ttgcctctga aaccagcatt ttatggtaat agttccactg ggttaaattc atgttccctt  3661 taagtgaagt ttaaaagata cctaacttct tctttgaaat ttgtttgtgc ttctgaggaa  3721 gagtgcttgc agcagagctc agtttactag agtttttcat agggaaaaaa agggagatgc  3781 atggtgttgt tcattattca gttaatattt ttctctttcc aaagttagaa caagagaaag  3841 ttttcaattt ttataagcta tgctagttca gaagtgggtt ttatgttatc aagtttcttt  3901 gttaatctca aaatgaaatg ttgttttgct tttcttagat aatgaaacag accagatttt  3961 acttgcaggt tgatgtgtaa gtccttgcct tccacctctt caactcattg tgtgagaggc  4021 attttgtctt tagtcattgt tttaaaaaat aaaagtgaaa tgacataaca aaaaattaac  4081 cattttaaag tgaacaattt ataaagtggc atttagtatg ctcacaatac tgtacaatca  4141 ccacctttgg tttcaaaaca ttttccctac ccctaaaaga aagccagtgc ccattaagtg  4201 attattcccc tcagcctctc tcctggtaat catcagtcta ctttctgttc ctatacagaa  4261 tatttcatat tagaggaatc atacaatatg tggcttttta tgtctagctt ctttcattta  4321 gcatgatgtt ttctaggttt gtttatgtag caatacttca ttccttttca tggttgaata  4381 atattctgta attgtatgta tataccacaa cttgtttatt catccattaa ttattcagtc  4441 tttttttgtt aaatatctaa acattctaaa accagtgtat tcatttatgc agtgaacatt  4501 tgtggagcat attatgtatc aagcagtgtg ttggatccca aggatgtaaa aatgaccgtc  4561 atttataata ataatgtgat acatgctgtg gtggagatgt aaacagtgtt tatatgactt  4621 gaagaagtgg ttaattcttt ttggcatgga gattgaattc attttgcaag cagatttttg  4681 ctgatggcaa agagaagagt gtctagatat attttgtcca ctgtagctgt agatcagtat  4741 atctgaaatg gtgtaagcta tttgaggaca gtgctcttta ttgttttcta ctgtaagtca  4801 ctaacaattt tggctgtttt atttctagac tgtttagtct tttgttaaat attgccaagg  4861 aggggctggt cactgttctc gtaaactagt tccttagtct gtcttaagaa tagactgaaa  4921 tgcagatgat aagtagtcta gaggaaaaga gaggctttag agattggttt cggctatacc  4981 tatcacaaga tttcgattgg tcagatggct atgtctgggt tggattcaga gtgtgttagc  5041 agaacacagc catgaactac cactgcaagt ttctttgagg ccagcctact ttctgagaga  5101 gaggcaattc tttgtacaca tactattctc ctttgtcagt cttattctgt taacttcagc  5161 gataaggcat gactctgtgt gcagcagctg ttaataattg gtaaatgggc tgggtgcggt  5221 ggctcatgcc tataatccaa gcactttggg aggtcgaggc aggcgaatca cgaggtcagg  5281 agatggagac tatcctggct aacatggtga aaccccgtct ctactaaaaa tagaaaaaaa  5341 attagccggg tgtggtgatg ggcgcttgta gtcccagcta cttaggaggc tgaggcagga  5401 gaatggcttg aacccgggag gcggagcttg ccatgagccg agatggcacc actgcactcc  5461 agcctgggcg acagagctag actccgtctc aaaaaaaaaa tttggtaaat gggtttgagg  5521 tatagagctg gacattgttg gagaaggact atggctagaa ctatagaaat aacgtacttg  5581 ctagaagaat gtgcttgaga catcagtgga attttttatt tttcagccta caacacagca  5641 gtcccctcag gatgagcagg aaaagctctt ggatgaagcc atacaggctg tgaaggtcca  5701 gtcattccaa atgaagagat gcctggtaag aatggagatg tgggaggcac agttgcagtt  5761 cgtgtgttcc taaggaagca tgtgcagtgt cttctagagt caggtgtttc tggtaaatct  5821 aatcttcacc gtttaccagc atctatcttc agtctcatct ccctcaagca ctttgtggag  5881 caatttcaac aaagagccct gtttactcac atgtatattt atggtttggg attgtctgtc  5941 ttccctacta gaatacaagc tcataagaat aagagaccct tccttttatt tacacattac  6001 tgtattatta ccacaccagt gtctgaccag aattactagc ctccttggtt ctatacctca  6061 gacctgagga atatttaaca tataataggt actcagtaaa tatttgttga atgaatggat  6121 ttaaatgctt tgcatttgaa ttattcagct ttttttctaa atatcttgaa aactttaatt  6181 tctttgctga atagatatat ttattgtaga agctagctta aaaattatac ttaacactta  6241 tttacatatt tttatattct aaaagataaa gtaagagata atctgtgtag atacttttga  6301 ttctctggat taaaatgtaa ggaattgagc caaattggtt agtactttaa actataaatt  6361 actgtgatga agatgatgct attttacctt tgtaaaatgt cttactgtgc tttctaaagc  6421 atagtaatat gctcttgtgt cttttattgg tttaattcct aacaaattgg gaatgaaaaa  6481 taaatgtctt ggaatggaga agctgggttt gctattgctt gcttctttct cttcctgtgt  6541 atggatagtg tttcctctat ctcaaggaat tgcttgcatt tctgagttaa gtggaacata  6601 tgggcattgt gagggcttga agaatgcaag aggaaagcaa acttacatgg atagtcattt  6661 cagacagctc tgaagagtct ttaacccatg acaaagccat gtcaggatag tatcttcctt  6721 cacctgaatc agtatgccag ttctcttgat tgcaggtaaa atgtgatgaa tggagctagt  6781 ttcctagtct ctatagattg aaaagattag cattctatca agaagcttgc agtcttagct  6841 atgttaagtc ttactaagaa tcatgtatct ttttcttttt cagtagagac ggcaaggtga  6901 accgatctaa gttgtttttt taatgtggtt aaaatcattt aagtgcggta ttcttttaaa  6961 actatgtaac aagtccttga tgtaaagaat ttgtacaacc aagataaatg tttatttaaa  7021 ttaagcattc tcatctattc tcttggtatt tctgtaggac aaaaacaagc ttatggatgc  7081 tctaaaacat gcttctaata tgcttggtga actccggact tctatgttat caccaaagag  7141 ttactatgaa ctttgtatct tttgaatgtt gaagactaaa catttggacc ataccttttt  7201 cttgataagg cctattttgt ttgttcttta tgaagttttt ctggagttat cttattcttc  7261 gttatctgag tcacatggca ctccttctcc atgcagatgt gctaagtgag aaaaacactt  7321 tgagagtact cctttcctat gcttaaacat ctttaaatgt gttgtcggtg catctcaatt  7381 ttcagaccct tcatgaggat atttaggcta tgacacagtt ggttctttaa tacttagatt  7441 ttgttatgca gcagtctcaa atggacagga atttaatcat ttgccatttc aaaacccatt  7501 agcagtctga caggtaacca ttgtatttac tgctttgctt gaccacacat gctttaaaac  7561 ccttatttta aagtaagaaa agtccggcta aaattcatcc ttcgcttgaa cactttctta  7621 aaggactaaa acttaagatg tctgcccagt agttagtaat gactccaaca agtttcaaag  7681 ttttgtttag gttggcttat ttttattttt agtccttaat cataattaaa agatatggcc  7741 atttctgatg aactgcacta cttggaggtc tacctgacag atgagtttgc taaaggaagg  7801 aaagtggcag atctctacga acttgtacag tatgctggaa acattatccc aaggctgtaa  7861 gtaattacaa atcagagaac ttttgtgtct gtatttctca ctatatgtta cgtcttttat  7921 gattatcagc ttaagaaaaa gttttaaggg taacttctta acaaattgag atgaacattt  7981 tggtagatat tctcttactt gttttagagt aactagattt acgttttatg tagatatttg  8041 aggaattttg gaaatagaaa aaatggacat gcttgctatt ttttttaatg tcttgactat  8101 tagaaaaatt aatataattg ttctcttcct aatatgttta aaggtaatat ctatgttgta  8161 tatatacagt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt atagtttttt tagaggtcag  8221 tcagtggtta tattttaaat gagatatttt ccttgtcatg cgggagaaaa caacatggtt  8281 cctgtcttgt ttatttaatg ttttgttcag tgtgtttgga aataaattct tgatttgaat  8341 attttatttc taatcagcat ttcttcataa ttttcctagt taccttttga tcacagttgg  8401 agttgtatat gtcaagtcat ttcctcagtc caggaaggat attttgaaag atttggtaga  8461 aatgtgccgt ggtgtgcaac atcccttgag gggtctgttt cttcgaaatt accttcttca  8521 gtgtaccaga aatatcttac ctgatgaagg agagccaaca gagtaagtga ttttctttct  8581 taattttgtt gcaatatttc tttcattgta gaatgtataa aagtgtggaa acatatacag  8641 aaacaaagtg tgaataattc ttccacccag tcagccattt aggtagcatt tgtatataga  8701 tttcctttgt aatatagaac tcctcagtat atgtggtatc atctaaaatg tactcttatg  8761 caaattttat ctttggattg ttaggacctg cttttttcat ttaatgtaat tttttctact  8821 acattaaatc ttctttgaaa ataaaacttt tttaagagag ttgtatttgg aaattgaatt  8881 tgtaatgaaa taataaagtg tgagccagct ggatttcata attgttcctt tagtgtctat  8941 cagtttttat aatttataga ctgctagtta ccttggaata taagtgattt gaattatctg  9001 ttacgagtta gctattaact ccagagaagg aaaaataaaa gccattcaga gacactcctg  9061 tctcttgtgt tatcagtatt ctagcatcaa agtctactgt acttttatcc cacagcaggg  9121 gcagatggtc agccaactgt ggtcttcagt ggggtgagct gttcacatga caggtcccca  9181 gattaaagaa cttcattcct tttttaaaaa gtttattcat ttattttctt tcttttttta  9241 atttttaatc ttttttcagt ttgccccaac agattttgtt tttttctttt taatattttc  9301 atttatttct aaggttttta atatatcatt tatttctaat gttttttaat atatctgcat  9361 caatttcttt taaaacagta cagaaaagat aaaacattta acaatgtaga gaaattgatg  9421 aagttacttg ctttattatg ttttgagtgt ccgttttgag catttaatta ggcaatcaat  9481 aacaattttt gaaaggtact gaggtctcca tcctaggaga cgtagaaaaa taaagcagga  9541 aatccatggt ctcttccctc acaaagctta cattccaatt aaaaacaaaa tattcaacag  9601 taaaatgatg tagttagcag tacaccataa gtgttacatt ttttagcctt ttgtttttgt  9661 ttttggtttg tggggatggg gtctcattat gttgcccagg ccagttttaa actcctggcc  9721 taaagcgatc ttcctgcctt ggcctcccaa agcactggat tacggacatg agccaccatg  9781 cccatccttc tagccttttt caattaagga agttgccata agagcaagtc cagttggccc  9841 aggatgagga gttggggaaa gtaatttgcc ctttaaattt atactgtcct ctccatggta  9901 ctttttaccc tagagtctgt tccctttgaa atttaacact aagccaatga agttgaaagt  9961 gattttttat aaagcattgg tgtactatag agataagtag gaaatacaca aaggagaagg 10021 atagtagtaa gttggtcctg taaatactgt gtaaagactt ttctgtttct ttgcagtgaa 10081 gaaacaactg gtgacatcag tgattccatg gattttgtac tgctcaactt tgcagaaatg 10141 aacaagctct gggtgcgaat gcagcatcag ggacatagcc gagatagaga aaaaagagaa 10201 cgagaaagac aagaactgag aattttagtg ggaacaaatt tggtgcgcct cagtcagttg 10261 gaaggtgtaa atgtggaacg ttacaaacag gtttatatat ttttgttacc tcttcttatg 10321 ttcagagata aactgaaatc tgatttttaa aatcagaata tttttgttat acaatagtac 10381 attgaaaaac atcttaaaat ggctgttatt gaagaagact taaacggaaa gatatatatg 10441 cagtgtttgt ggattggaag acttaatatt gtcaaaatag catttaaaaa gaattgattt 10501 atagatccaa tgtaatctca gtcaaaatcc cagcagactt ttgtagaaat taagaagctg 10561 attctaaagt ttatatgaag aaacaaagaa cctggaacag ctacaacgaa tttgaaaagg 10621 aagaacaagt tgaaagaccc aagcaacctg aattaatgat ttactctaaa gctgcagtga 10681 gatcctgtgt ggtattggtg aaaaggatag acacacaaat caatggaggg gaataaaaca 10741 gggaatggca aactttacct gtgagggacc agataataaa tggtttttgg ctttgtaaga 10801 catgtgctgt acaacaagct tgtccaacct gcagcccagg acagccttga atatggccca 10861 acataagttt gtaaacttta aaacatgaga ttttttgctt tttttttttt tttttttttt 10921 tttttttaaa gctcatcagc taagtgtatt ttatgtatgg cccaagacaa ttctaattct 10981 tcttcaagcc aaaagattgg acaccctagt ctacaactaa taacagtgca gatatggtgc 11041 aaaagcaccc acaggcaata tggaagtgaa tgggcatggc tgtgttctag taaaacttta 11101 tttgtaaaaa caagcagcag ctcagtttac cgatctctga ctggacaatc cataatagac 11161 ccagatattt atgatcagtt atttttgata aaagtacaaa ggcaaccttt tcagcaagtg 11221 attctggaac aattggatgt ttatatgcaa acaaaaaacc ctgaaccttg acccatccct 11281 cataccatat agaaaaaaca cagaaatcaa tcagagacct aaatatagaa cctaataatg 11341 ttagaagaaa acacagagga aatctttatg acctaggatt agacaaagat ttctgaggat 11401 atacaagcac aagccatgaa gaaaaaagct cacttttgag aggccaaggc agatggatca 11461 cttgagtcca ggagtttgag acaggcctgg gcaacatagg gagaccccat ctctacaaaa 11521 attaccaaaa ttagctgggc atggtggaac gtacctgtag tcccagcact caggaggctt 11581 gaggtgggag gatgacttga gcctaggagg tggatgttgc attgagtgga gattgtgcca 11641 cttcactcca gcctgggcaa ccgaacaaga ccttgtctca aaaagaaaaa agcttttaaa 11701 gtttagaagt gaagtcttgg tgagaaaaat ctcaaatacg attttcaagt tagtagttca 11761 aatgcgttac tagaggaata gcttaagatt ttgaaaacag attttaaccc ttatgtgtgt 11821 tttttctctt ttagattgtt ttgactggca tattggagca agttgtaaac tgtagggatg 11881 ctttggctca agaatatctc atggagtgta ttattcaggt agctgggaac atttcatttt 11941 tttttaaacg acctatttta tctttcatta aatttaattg ttttgaaaaa attttgatgg 12001 aataggaaat aagctttcct gaataaagag ttttccttgc ggggtgtggt gactcacacc 12061 tgtaatctca gcagtttggg agttcaaggt gggaggatct cttgaggcca ggagttcaaa 12121 accagcctgg gcaacatagc acgatgccgt ttctataaaa aattaaaaaa atttttttag 12181 tgtttctttt ttttttcatg taatcttgct tcttctaaaa ataatttaaa aataggaatt 12241 ttctgtttct aacttatacc ttggtctttg tatcaatgtg gtttgttttc ctccaaaatg 12301 taggaatgag taatctgagt tttctaggtc tctgtagctt tagtttaatt gtaggtgcac 12361 tttgtttatt ggaatatttc tgtctgagct tatgtttagt agagaggttc aaaagtaatg 12421 tgtttgaatt tagttgtata agaatacagt gtttttttcc cacaaatgtg aactttacca 12481 tatgtgagtc cagaatatta cgtgaaatac ttttatttgt attgatcatt tgattttcag 12541 gttttccctg atgaatttca cctccagact ttgaatcctt ttcttcgggc ctgtgctgag 12601 ttacaccaga atgtaaatgt gaagaacata atcattgctt taattgatag gtaagacctt 12661 ccaacactgg cggataaatg ctctgacttg ggaataatga attttaaaca tttttttgaa 12721 ttatttgttt ctgttacatc tttatcatac caatgatctt aatttaatta tactataaat 12781 aatttagctt tgtgagtatg agtactaggt acttgtctag gttagacatg aaagaggctt 12841 aacttaaatg tgcaggagac gtgaagataa tgaatatctt tattctgtgt gcttaattga 12901 catttaaaga tgttgtacag acttattttt taaatcatac aaatccaaag atcatattga 12961 agaacaaaat ttgtttttta ccatgatgta agtatcttgc agtgggaact catttgattt 13021 agagtagccg taagatactg atgattgaaa atgttcaagt aatcactcta tcatcacatt 13081 ttcttaaaga aaaaatttta agtatcaaat atgtttagta catccacttt tttattttct 13141 taggtttttt tttttttttt tttttttgag acagatcctc actcttgtca cccaagctag 13201 agtgcagtga cgctgtctcg gctcactgca acctctgact cctaggttca agtgattcta 13261 gtgtctcagc ctccggagta gctgggatta cagacatgca ccaacaagcc cagctaattt 13321 ttgtattttt agtagagaca gggttttgcc aggttggcta ggctagtctc aaactcctga 13381 gctcaaatga tctgcctgcc tcagcttccc aaagtgctgg gattacagac atgagccact 13441 gcgcttggcc aatgggtggc ttttttgcag ccatgttatg tagtagtata tgatgtctgt 13501 cctacacttg taagcattgt catgaaacca gaaacctaag agaagattta tttctgcaga 13561 taccttttgt atgtttttta aaaaactaag ttattagttt taaagtctga gaatttagat 13621 aacaaatttt tccaaattgt cagctcaatc ctgggcagca aaaattccat acttattggg 13681 cccactctta aaggaagcta gtaactggat tttcctgagt tgcctgtaat gtcacttaca 13741 catctctgtc agtagtgatg cttctgggca tagcaaaatg tggatgtagt tgtgactgac 13801 aaacagataa tgataatgaa acatactatt ttgagtaatt taagatgtgg gaaataaaag 13861 ttaattttat gaattttaga cttagttgta tttcaagctt tagtaaaaat gcagtatctt 13921 aaaatagtct atgtactttt attttttaaa ggttatttat ttaaatcatg gttgttgaat 13981 acatttgtca ctttaatgca tttctgtcca tatctgctta attatgcttc aaagagttga 14041 gagaattatc ttgttgaaaa tctacttaat atggtgtgaa ataagaatgc tgatgaaaaa 14101 ggtttcattg gcaaaactgt ttagttaaaa atgaattgag gaggccgggt gcagtggctc 14161 acatctgtat tcccagcact ttgggaggcc aaggagggag gcttgcttga gtccaggtca 14221 gtaccaccct gggcaacatg gtgaaacccc atcactacag aaaacacaaa aattagctgg 14281 gtatggtggc acatgctgtt agccccagct actcaggagg ctgaggtgga aggaggatag 14341 cctgagctca gcaggtggag gtttcattga gtggagagtg cgtgactgca ctccagcctg 14401 ggcgacagag cgagactctg tctcaaaaca aaacaaaaca aaacaaaaaa aacaaaaacc 14461 ttttgggctc atacaaaata tagaaaagca ataaagaata agatgtcatc catgatctca 14521 ctacccaaac cctgtatctt ttaaaataaa ggggtgtttt tttttttttt agattagctt 14581 tatttgctca ccgtgaagat ggacctggaa tcccagcgga tattaaactt tttgatatat 14641 tttcacagca ggtggctaca gtgatacagg tttgtgtagc atttctccta agttctcaaa 14701 actttgaaac ttctctgcct tccttttaca attgtttaaa ataaattgtg tggttttcta 14761 aacattccag tctagacaag acatgccttc agaggatgtt gtatctttac aagtctctct 14821 gattaatctt gccatgaaat gttaccctga tcgtgtggac tatgttgata aagttctaga 14881 aacaacagtg gagatattca ataagctcaa ccttgaacag taagtcagtt acatttttgt 14941 aaaaatcctc aaagatattt ttgtcctaga tttgcttttc tttctcaatt gttttttgaa 15001 ctgctggcat ttgtcttgtt ttaatcatgc attaagattg tcatgcttag cactactagg 15061 ggcagaaagt agtgaccaat tacttgtttt tttatattaa ggaaattgtg gtacctatgg 15121 accataggca gtcttcaggg accagtgtct ccaatttgga tccctttctg tgtgtcaggg 15181 gcatccaatc ttttggcttc cctgggctgc actggaagaa gcattttctt gggccacaca 15241 taaaatacac taacactaac aatagctgat gagcttaaaa aaaaaatccc aaaaaaactc 15301 ataatgtttt aagaaagttt acgaatttgt gttgggccgc attcaaagcc atcctgggct 15361 gcatgcggcc tgtgggcttt gggttggaca agcttgcatg tgactgagtt tgttcttaaa 15421 ctggtaagga aactttgtca ggcagtattt atttccataa gtggtgtttt cctacgaatc 15481 agcacatggt gaaaaatgag gggctatgta tatttaaggt gcagaattaa attggtttaa 15541 atatcttttc tattttgagc tttgattttg ataccttaaa ggaaatatca acagtactat 15601 ttccaacctg aagcctcctc agctgttctg tcctagactt atggcgtcct ctagtggcca 15661 ctatgggcag ctatgatcct gttaccttcc ccagcagttc ccttcctgcc ctgttcccca 15721 ctgctctggc ttgggtcaag ccaggcctgc ctcccgccaa catattcttc agaattttac 15781 ctcatgtaat cttcctcctt tctatctccc ttccagtggt ttacctgcat caagaaaatt 15841 tcttcttttt ttcctccctt tgtgttaccc ttgttctttt ggtcattttt ggttttgtgt 15901 gtgtgcaaac tgaaaacaag tccagatgtg gaatgataag tgtgagagaa aattaaatga 15961 tgtgccaggt gtggtggctt gcacctgtaa tcccagctat tcaggaggct gaattgggag 16021 aatcacttga gtccatgagt ttgagaacag cctgggcaac atagcgagac cccgtctcta 16081 ataaaaaata aaattaaaaa taaaaaaaat ttaaattaaa aaaactaaat gatgtatctg 16141 tgtctttctc cccaagtgaa ttttaaagta aaaatagaca aagtaattag aaataacaac 16201 ctctaaagag gttgtaataa atgccccaat atgcctcaat atctacagaa tgattttact 16261 aacaactacg taaaagtcag tcagcctgct tttccttaat caccaacatc tgatgcagaa 16321 gaaatagttt atgtgttttt ctgttgtgtc aaattgctgg ttttgcatgg agtttttttc 16381 ctatttattt tcatcatgaa tatacaatac ttgttggctg gcccctggga accaaactac 16441 cacttaaaat acttccctta gaaatgtcat caaattctag acagtcatct taactccagc 16501 tataccatct gttcatgagt tggaaactgt atctagtttt gtatcaacag aaaaataata 16561 gatgaatata tatttgtgtt tagataagca tttttatcct cctgaaagga ggttgttata 16621 gtcttctgtg gtggtatgat tcacttgacc catttccttt aatgtgtaat gaaaaatttc 16681 aaattcttat ggaacaaatg ctatttgtgt atatagaaag ttaattttat tcattaagac 16741 ttctgttttt ctttttgtag tattgctacc agtagtgcag tttcaaagga actcaccaga 16801 cttttgaaaa taccagttga cacttacaac aatattttaa cagtcttgaa attaaaacat 16861 tttcacccac tctttgagta ctttgactac gagtccagaa agagcatgag ttgttatgtg 16921 cttagtaatg ttctggatta taacacagaa attgtctctc aagaccaggt aagagaatac 16981 ctacgtgcta ttttagggaa acagtgttac aattttagac tttggaccta gatacctgag 17041 atgggagggg agggtaattc aatactaaat aaaatttaca agtaactttt tcattatata 17101 aattaaaaat tggagatgta taaagaatta taaaacattt ataattccac cagatagaga 17161 ataaccactg ttaattaaca tttggtgcat atctttccag acttttgtct gtatatgtgt 17221 gtatgacata catgtgtatc gactttctca ccaaaaaaag gaatatcttg ttgatactgt 17281 attgtaattt tataactgga aacacttttg ataatggctt tgtatgccaa tggtttcacc 17341 tcagtgggtt tcttgtgcct cgcatgttac aggtggattc cataatgaat ttggtatcca 17401 cgttgattca agatcagcca gatcaacctg tagaagaccc tgatccagaa gattttgctg 17461 atgagcagag ccttgtgggc cgcttcattc atctgctgcg ctctgaggac cctgaccagc 17521 agtacttggt atgagtttac ccttagtata tccctgtatc agctcctagt gaaatcacat 17581 gttcaagtgc ttaaaatggt ttaattcact ttctggtctt agatggtttt gaaggaattg 17641 caactgaatt aaagattcac ttgaacctgg gaggcggagg ttgtagtgag ccgagattgt 17701 gccactgcac tccagcctgg gcaacacagc gagactccat ctcgaaaaaa aaaaagattc 17761 atggcatcca tgggctttta ctttatatat aaacacataa ttgtttgtaa acttctggag 17821 catgtgagta acaattcagt tgctctgatt tcttttgaag actctctgag aattacaaaa 17881 aagtctgtct tcttttgctt gagtgccgat aattattcca tgttcatttt ttctgaacta 17941 tgtattgctt ataataaact ttataagaaa tacaattctt atatttaatt ttacttttcc 18001 aaatttgcaa gtataaatta tatttgtcat attgaaaatg tgagtttttg ttttttgatg 18061 aaagatttaa aaattcattt tgcctttttc ttaacttttt tttttctgat aaagaacaat 18121 cacatgaggt tctctcttta ttattagtcc acagggaatc attgtgaaat ggataaaaca 18181 tgttgcctga gtaggtgtat cagtgaccga tactagatag atagtttatt ttagtgaagg 18241 gttagcacag ttggctgctt aattattgtt tgggcaaagt agtttaacca ttcttggatg 18301 cataaggcta ttaggctgct atgatgaaaa agacatttgc ttgaggatgt cctgactgtc 18361 tcatcccttt ctgttgactt tcttcattgt agttgacaca cctgtacttc ataatcagtg 18421 tgaaataaga ggctgacttc tgttgatagt gtgatggtct ttgtcttggt ttagtgacaa 18481 acattccagg actgtggtat tgtgctctgt gagctatgtg atctgtacag agtgactgtc 18541 ttaagtattt taactgattg ccttatgttt ctgtgtgaga ttgtttgtat ctgtgtgttt 18601 tcattttcta ttgcctacca aatatagtag ttagaaacta ttccttccgg cggggcatgg 18661 tggctcgcat ctataatcct ggcactttga gaggctgagg tggatggatc acctgaggtc 18721 aggagttcag gatcagcatg gccaacatgg tgaaacccca tctctactaa aaatgcaaaa 18781 aattagctgg gcgtggtggt gggcgcctgt aatcctagct gctcaggaga ctgaggcagg 18841 agaatcactt gaacctggga ggtggaggtt gcagtgagtg gatatcatgc cattgcactc 18901 cagcctgggc aacaagagtg aaactccgtc tcaaaaaaaa ttatgccttc tgcatgtggc 18961 tgattggtta ttcccatgta tggagatctt taatgatagg gtcattagct ctgactgccc 19021 ctaggggaaa tgcattctct tattcatcta ccatatcagg aatttcacaa aacctgaatg 19081 ccattgtgtc acatatacta aaaatatttt ataaactctg tgtttttctt gtaatttttc 19141 tgaattggct atatgttgtg ccatttcaga aaaaaaaatc caagaaaaac acagaattca 19201 tggaatattt cacaagtagc tcttttaaag tatgttagca ttttccttga cttaaatggt 19261 cttaaaattt ttttgaatga ggaggtatga tgtaccagta atatgcatat agttgttgtg 19321 tatcatagta atagttaata ttactgagct tatgccttgt gctaagtagt ggtaagcctt 19381 cacatgtgtc acttgatctt cccaacaacc ctaggagttt atagaaactt gtggctaaga 19441 gaaggtaaat aatttgccca aggccacaca tgtaataagt attagcatct gcttttaaat 19501 gtgagtctct gagtatcttc acagccttct ttttttctct tttctttttt cttttttttt 19561 ttttttgaga tggaatcttg ctctgtcacc caggctggag tgcagtggca tgatcccagc 19621 ttactgcaac ctccatctcc tgggttcaag caattctcct gcctcagcct cctcagtagc 19681 tgagattacg ggtgtgcgcc accatgccca gctaattttt gtatttttag tagatacagg 19741 gtttccctat gttggccagg ctggtctcga actcctgacc tcaactgatc tgtccacctt 19801 cggcctccca aagtgctggg attacagaca tgagtcacca cacctggcca gagcctacat 19861 tctttatcag tgcagcatac tttgcacatg tgtgtatgaa aatatattta aatatatctt 19921 tgcttctaac tcgctacctt gggcaggtta tacaacctct ctgaaactca ggcttcccca 19981 tttgtgaaat ggaatagtat ctgtctctgg gttgttgtga caacttgagg agataagaaa 20041 tatgtaaatt gcctaccata aagtatggta cattgtatat attcacaaaa tgttagcaat 20101 gatgattaga gcccacattt atttcacaaa tgattaatca gagtttggaa attttttttt 20161 ctttaatgct tttgggtcag attttgaaca cagcacgaaa acattttgga gctggtggaa 20221 atcagcggat tcgcttcaca ctgccacctt tggtatttgc agcttaccag ctggcttttc 20281 gatataaaga gaattctaaa gtggtgagtt tacttttaag tatttaggta ctttttttcc 20341 tctttcatca ctctgagtgt gtgtgtgttt gttttatatt ataaaaaatt tcaaacgtac 20401 aaaaatagac agtggtataa taaaatccca ttttcgccaa ctctctattt gttacttatc 20461 ctgtgctaag tgttcctaac ggtgatggtg gtggatcaca tactgaggga tcacgtaaaa 20521 agcacttaga aatgcaagat taaagcagtg tgaatttatg ctgaaactct ttcctaagtt 20581 ctaattcagg ttagctttaa aacctaagga gagggcctag cattgcagtc gtttctctct 20641 aaaggcatat cattgaataa tatgagttgt gggcaacttt ttatgagctt ttttcttcct 20701 caaaatggaa ccatggcttg agtcttcaca gtgtagtttt gaagaaaata cctcaagctc 20761 acgtacctga aagttggaca ttcaggttaa tgttaaggaa caacctcagt aacttaattt 20821 tgtttgtttg tttgttttga gatagggtct cattctgtcg cccgggctgg agtacagtgg 20881 cgcagtctta gctcactgca acctccaact cctgggttca agcgattctt gtgtgtcagc 20941 ctcctaagta gctgggatta caggtgtgca ccaccatgcc cagctaattt ttgtattttt 21001 agtagagaca gggtcttgcc atgttgacca gttggtctcg aacttgtggc ctcaggtgat 21061 cctgctgcct cagcctctca aagtgctagg attgtaggtg tgaaccactg catctggcct 21121 cagtatggac ttgattttct cgtaatagag aaaaaagatg tatgcagtag acctaccagc 21181 atgaaacagc agcttttggc caatttttat taggccagct tatcattcac tctttaccag 21241 cgtttatgga taggaatttg tgaatataac aataaaaata gcaaccagcc tacattacaa 21301 agccatagta attaaagcag tatggtaata tggtactggc ataaaaacag acacatagac 21361 caatggaaca gaatagagag tctagaaata aacccacaca tatgcaataa actaatcttt 21421 gataaggaca ccaagaatac acaaagggga aaagaatggt ctcttcaata aatagtattg 21481 ggaaagttgg atatccacat gcaaaagaac gcatttggac tctcatctta tgccatatat 21541 aataatgaac tcaaaatgga ttaaagacct gaaaccataa agctcctaga agaaaacata 21601 gggaaaaacc tccttgtcat tggtcaatga ttttttggat atgaaaccaa aaacctatgc 21661 aactaaagca aaaataagtt taaaaataag caaaaaataa gtttaaaata agcttaaaat 21721 aagcaaaaat aagtttaaaa taagcaaaaa ataagcaaaa ataagtttaa taaactaaaa 21781 accttctgta caacaaagga aacaatcagc agagtgaaga gacaggcaat ggaatggggg 21841 agaatatttg caaactatac atctgaaaag tggtcaatat ctaaaatata tatggaatgc 21901 aactcaatag caagcaaatg aataacttga tttaaaaatg agcaaaggat ctgaatagac 21961 atttttccaa agaagacata caggtggcca actggtatat gaacagatgt tcaacatcat 22021 ttatcaggaa aatgtaaatc aaaaccacta tgagatgtca cctcacatct gtcagaataa 22081 ctgttatcaa aaaaacagaa aatcaagtgt tggcaaggat gtagagaaat gggaaccctg 22141 tttattattg gtgggaatat aaattagtat agccattatg gaaaacagta tggaggttcc 22201 tcaaaaaact gaaactagaa ctaccatgtg accctgcagt cccacatcta gttatgcatt 22261 caaaggaaag gaaatcagta tctcaaagag atatctgcac tcccatgttt attgcagcat 22321 tattcacaat ggctgagata tggaaacaac cttagtgtcc atcgatagat gagtaaagaa 22381 attgtgttgt gtatatatgt gtgtgtatat acgtatatat gtgtgtatat gtatgtacgc 22441 acatattctc tacatagtag aataatactc agctatagaa atgaagaaaa tcttgccatt 22501 tatgacaaca gggattaatc tggaggacat tgttctaagt gaaataagcc aaacacagaa 22561 aggcaaatac tatatgactt catttatatg tagaattgtt ttttaagttg aattcatcca 22621 gcctgggtaa tatagcaaga cccaatctct attaaaaaat aaaaaggcca ggtgtggggg 22681 ctcacgcctg taatcgcagc actttgggag gccgaagcag gcggttcacc tgaggtcggg 22741 agtttgagaa cagcctgacc aacatggaga aaccccgtct ctactgaaaa tacaaaatta 22801 gctgggcgtg gtggcgcatg cctgtagtcc cagctactcg ggagtctgag gcaggagaat 22861 cacttgaacc cgggacgcag aggttgtggt gagccgagat cgtgccattg cacttcagcc 22921 tgggcaacaa gagtgaaact ccgtctaaaa aaaaaaaaaa aaaattaaaa aattagccag 22981 gcgtggtggt acatgcctat agtcctagct actcaagagg ctagggcagg gctgggtgtg 23041 gtggctcaca cctgtcatcc tagcactttg ggaggccaag gtgggtggat catttgggat 23101 caggagtttg agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa 23161 aattagccag gtgtgagggc acatgcctgt aatcccagct acttgggaag ctgaggcagg 23221 agaatcactt gaacccggga ggcagaggtt gcagtaagct gagatcgcgc cactgcactc 23281 cagcctgggt gacaaagtga gactctgtct caaaaaaaag aggctagggc aggaggacag 23341 cttgagccca ggagttggag gctgcagtga gttatgattg tgcttttgcg ctccagcctg 23401 ggtgacagag ggagacccag tcactaaaaa atggttgaac ttgtgtaagc aaagactaga 23461 acagtagttg ccaaagaata caaactttca gttataagat aaaaaaattc tggggatcaa 23521 aacgatttag ggcaaataaa taaaagtaac tagcctttac ttatttacta gcatttctta 23581 ctgtgttgtc acccactgtg ccaaggtcta tgactgccac tgtcactttt tttttttttt 23641 ttgagtcaag gtctttgttg cccagactgg gatacagtgg tatgattacg gctcactgca 23701 gcttcgaact cttaggctca agcgatcctt ccatttcagc ctcctgagta gctgggactg 23761 caagcatgtg ccaccacact ggctaatttt ttattttttg tagagacaga gtctcaccat 23821 gttgcctagg ctggtctgaa actcttgggc tcaagcgatc ctcctgcctc cttggcctcc 23881 ctaagtgttg ggattacagg catgagccac catgcccagc ctgtcgccat cttttaaaaa 23941 tgaaaagaac tgattgcttt aacaagaaga aatttggata gtcaatcatg ataaaatatt 24001 taacctcgct tgtaattaca acagcgaacc ttttaagaaa tcaaattggc aaagagaaat 24061 gaaaaataag gttcagcaat ggcgatggtg tgatgaaaat tcattctcat ctaatgttgg 24121 cagtgtgaat tactataata cttctaggaa gttggcggtg tgtaagtagg gtgttaaaat 24181 attcaataat tttacttcca agtggaattc caagaattta tactaaggga ataattaggg 24241 tctcaataaa gcttagtgta tatagaacat tcattgtaat attacagatt atgtctaaaa 24301 gggaatagtt caataaatta tgccatagcc agtctccata atattttcta gtcattaaaa 24361 tgatttcgaa ttagtatcgg gaagattgtt aggacaaaat aggaaaaatt agagctgggt 24421 gcagtggctc acgcctgtaa tcctagcact ttgggaggct gaggcaggcg gatcacctga 24481 ggtcgggagt ttgagaccag cctgaccaat atggagaaac cccgtctcta ctaaaaatac 24541 aaaattagct gggtgtggtg gcgcatgcct gtaatcccag ctattcggta ggctgaggca 24601 ggagaatcac ttgaacctgg gaggcggagg ttgtggcgag ctgagatcat gccattgcac 24661 tccagcctgg gcaacaagag ggaaactact tctcaaaaaa aaaaaaagaa aagaaaagaa 24721 aaattagata caaattactt gaagtgtgaa tcgattttaa ctctcaagaa aataaggtct 24781 agatgcagtg gctcacgcca gtaatcctag cactttggga ggctgagatg ggtggatcac 24841 ttgaggtcag cagttcaaga ctagcctggc cgataaggtg aaaccccttc tctactaaaa 24901 atacaaaaaa tagcagggcg tggtggcgcg cttgtaatcc cagctactca ggaggctgag 24961 gcagaagaat ggtttgaacc caagaggcag aggtggcagt gagccgagat cgcaccaaag 25021 agaaaaaaga aaaccacaca caaaaatgcc agttatatta cagttacata gaaaaaaaga 25081 aaggaagaca tttagcatcc gaatgttacc agtgattatc cgtgggtggt agatttaggg 25141 atgatgtgtg gatgattttg tgtatttttc taattttctc caatttggga atgtaactta 25201 caaatcagaa aaaacaatta tcagccaggt gtgatggctc atgcctgtaa tcccaacact 25261 ttgggaggct gaggcgggag gtttgctcgg ggccagtagt tcaagatcag cctgggcaac 25321 agaatgagac cctgtctcta caaaaaaaaa aaaaaaaaaa aaaaaaaaaa ttagccaggt 25381 gtggtgatgc aagcctgtag tcctagctat tcgggagtct gaggtgggag catcacttga 25441 acccaggagt tcaaggctgc aatgagctgt gatcacacca ctgcactcca gctgggtaac 25501 agagctgttg aaaaaaaaaa aaggaaagaa aaaacaggtt gagtgcagtg gctcacgcct 25561 gtgatcccag cactttggga ggctgaggcg ggcagatcac ttgaggtcag gagttaccag 25621 cctggccaac ttggtgaaac cccgccccac ttggtgaaac cccgccccta ctaaaaatac 25681 aaaattagct gggtgtggtg gtgggcacct gtaatcccag ctactcggga ggctgaggca 25741 ggagaatcac ttgaatccag gagacggagg ttgcagtgag ccaagattgt gccactgcac 25801 tccagcctgg gcaacaagag caaaactctg tctcaaaaag aaaaaacaaa taccaaatac 25861 attaacattg caaaggcaat ttaacctcaa atgatgtttt gagaagacat cctgcttgat 25921 ttacttgttt gccctataac tgaaacagag aaggaaaatg acaggaaaac tgtgcacaca 25981 acttacagta ttttgttcta ttaaaatgga tatcctggaa caagttaatt ttgaatttaa 26041 ggtaacttaa aatgtttttt cttgttttag gatgacaaat gggaaaagaa atgccagaag 26101 attttttcat ttgcccacca gactatcagt gctttgatca aagcagagct ggcagaattg 26161 cccttaagac tttttcttca aggagcacta gctgctgggg aaattggttt tgaaaatcat 26221 gagacagtcg catatgaatt catgtcccag gtgatgatct gttctttctg cgttgtcatg 26281 tcagctctgc tgggttcagt tgcttgtttg caggcatggt ggtaatgcac atgaatttac 26341 tcttctttta ctgaatgtgt aactaccacc ttcccaccat catggaacct gttaatatta 26401 ttgttgtaat tgactggtgt tgatcatttg ctgatgaaat ctaagatttc caagtgggtc 26461 atggtaaaaa tgtttcatgg aacataaaat tcgggaaatg cactcaattc ccaaaatcca 26521 gtttgggaac cctgggttaa acaaagttga aagaagtttc tttattgcaa ctttttagcg 26581 tttttaccat ctcagttgtg tcctgtggct ctcaagagag ggtgcagcat gttctgatat 26641 gaaggctgca gaagtctcac aggatggagg tttggtgaca agtactttgg aaaatgctca 26701 actagaggat ggttggtcct tgaaagtcct ttctgcttta tgttcactag gcattttctc 26761 tgtatgaaga tgaaatcagc gattccaaag cacagctagc tgccatcacc ttgatcattg 26821 gcacttttga aaggatgaag tgcttcagtg aagagaatca tgaacctctg aggactcagt 26881 gtgcccttgc tgcatccaaa cttctaaaga aacctgatca gggccgagct gtgagcacct 26941 gtgcacatct cttctggtct ggcagaaaca cggacaaaaa tggggaggag gtaaggtcat 27001 tcctgactgc atgatagcag acaggatcca taacagggat cagttgtcat ggccttgtgt 27061 tctggaggtg aaacatttgg ggtgcttgga aatctgatga acaaaattgc tttgttttgt 27121 taaaaaagag agtctcatcc tgtagtgaag cctctgcttt gaggatattg taacatagca 27181 agttcaaacc actacctgtt tttaaaaaaa tacagctgta tacttcaaaa caagaagaag 27241 gagaatgaaa aggatttaaa tttgttatgt ccctttaaaa cacgaaagag ccacggtagt 27301 gttgtgtttc tttgtatgaa aacgagatgt ttcattaatc tcttcactgt ccccctgccc 27361 ttttatttta gcttcacgga ggcaagaggg taatggagtg cctaaaaaaa gctctaaaaa 27421 tagcaaatca gtgcatggac ccctctctac aagtgcagct ttttatagaa attctgaaca 27481 gatatatcta tttttatgaa aaggaaaatg atgcggtaag tgaattagta aagtgttgtt 27541 aataaactaa tattttccct tcctactctt aggagatttg atatgtacaa aagtttatca 27601 ttctgatact ttaatcactg ttcatttgaa aaatgtaaaa taatttacag atgtcaaata 27661 ataggctaat ttgtcataat gttctagttt aagataattc ctaggctggg cgtggtggct 27721 catgcctgta atcccagcac tttgggaggc tgaggcaggc agatcacctg aggtcaggag 27781 tttgaaacca gcctggccaa cattgtgaaa ccccatctct actaaaaata caaaaattag 27841 ctaggcgtgg tggcaggcgc ctgtaatccc agctacttgg aagcctaagg caggagaatc 27901 gcttgaacct gggaggtgga ggctgcagtg agccaagact gtgccattgc actcctgcct 27961 gggcaacaag agtgaaactc cgtctcaaaa ataataataa taataattcc taaacgcagt 28021 atccttttag caatacagtt ttggtcaaga tttgtaagtt aaataaaatt ttgcttgttt 28081 ttcttttttt gacagagtct ggctctgtca cccaggctgg agtgcagtgg caatctcagc 28141 tcattgcaac ctctgcctcc caggttcaag caattctcat gcctcagcct cctgaatagc 28201 tggtattata ggcgcccggc accacgccca gctaattttt gtattattac tagagatggg 28261 gttccaccat gttggccagg ctggtctcaa aactcctgac ttcaagtgat ctgcccacct 28321 cagcctccca aagtgctggg agtacaggga tgagccactg agccgagcca attttgcttg 28381 ttttaaaggg ttgttttttt tttttttttt ttgatagtca gtaattgttc aaactaggaa 28441 ttgtatcccc atctttcttt tttcataatt actcaggtaa ttgatgagtg taacagaagc 28501 tcctcaaaac agttttatta aattgccttt cattttttgt ggtacgtgct tgatcatgaa 28561 tttgtacata ttcttttgta ggtaacaatt caggttttaa accagcttat ccaaaagatt 28621 cgagaagacc tcccgaatct tgaatccagt gaagaaacag agcagattaa caaacatttt 28681 cataacacac tggagcattt gcgcttgcgg cgggaatcac cagaatccga ggggccaatt 28741 tatgaaggtc tcatccttta aaaaggaaat agctcaccat actcctttcc atgtacatcc 28801 agtgagggtt ttattacgct aggtttccct tccatagatt gtgcctttca gaaatgctga 28861 ggtaggtttc ccatttctta cctgtgatgt gttttaccca gcacctccgg acactcacct 28921 tcaggacctt aataaaatta ttcacttggt aagtgttcaa gtctttctga tcaccccaag 28981 tagcatgact gatctgcaat ttaaaattcc tgtgatctgt aaaaaaaaaa aaaaaaaaaa 29041 aaacaaaacc cacaagcact tatcttggct actaatgaag ctctcctttt ttttgtttgt 29101 ttgtttgctt cattgttgat tgtgtatttt cttcattcct ggggagtact aacccaaaag 29161 cgtctgtctc ttgttttcta gtccagtttg agattaattt agaagaaagg aatactgtat 29221 gtgaaattca tcttgggctt tcccctaaat tgcaagataa ggccatgtgt aagattttcc 29281 ctaaaactag aatatattaa tgcatgtttg agaattttaa agcaccatgg tcaaaaccag 29341 aagctatatt ttgcatattt ggactcagcc atccattaag aacccatgtt gtcctctgga 29401 catatttatc aatataattg ggttttaaat agtataaaag aaaacttgtg atctatataa 29461 tttatgtatc accttcattg taaatttagc aggaaatgca tcacaattat gatttttttt 29521 tttgcaccag tgaaacaata aagatgctat taacaa

For example, the nucleotide sequence corresponding to the mRNA of the human VPS35 is depicted in SEQ ID NO: 13 (3298 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Sequence information related to LRRK2 VPS35 is accessible in public databases by GenBank Accession number NM_018206.4 (nucleotide).

SEQ ID NO: 13:    1 gctagagagg gcggggcttg gaggggccgc agcgtcacat gaccgcggga ggctacgcgc   61 ggggcgggtg ctgcttgctg caggctctgg ggagtcgcc a   tg cctacaac acagcagtcc  121 cctcaggatg agcaggaaaa gctcttggat gaagccatac aggctgtgaa ggtccagtca  181 ttccaaatga agagatgcct ggacaaaaac aagcttatgg atgctctaaa acatgcttct  241 aatatgcttg gtgaactccg gacttctatg ttatcaccaa agagttacta tgaactttat  301 atggccattt ctgatgaact gcactacttg gaggtctacc tgacagatga gtttgctaaa  361 ggaaggaaag tggcagatct ctacgaactt gtacagtatg ctggaaacat tatcccaagg  421 ctttaccttt tgatcacagt tggagttgta tatgtcaagt catttcctca gtccaggaag  481 gatattttga aagatttggt agaaatgtgc cgtggtgtgc aacatccctt gaggggtctg  541 tttcttcgaa attaccttct tcagtgtacc agaaatatct tacctgatga aggagagcca  601 acagatgaag aaacaactgg tgacatcagt gattccatgg attttgtact gctcaacttt  661 gcagaaatga acaagctctg ggtgcgaatg cagcatcagg gacatagccg agatagagaa  721 aaaagagaac gagaaagaca agaactgaga attttagtgg gaacaaattt ggtgcgcctc  781 agtcagttgg aaggtgtaaa tgtggaacgt tacaaacaga ttgttttgac tggcatattg  841 gagcaagttg taaactgtag ggatgctttg gctcaagaat atctcatgga gtgtattatt  901 caggttttcc ctgatgaatt tcacctccag actttgaatc cttttcttcg ggcctgtgct  961 gagttacacc agaatgtaaa tgtgaagaac ataatcattg ctttaattga tagattagct 1021 ttatttgctc accgtgaaga tggacctgga atcccagcgg atattaaact ttttgatata 1081 ttttcacagc aggtggctac agtgatacag tctagacaag acatgccttc agaggatgtt 1141 gtatctttac aagtctctct gattaatctt gccatgaaat gttaccctga tcgtgtggac 1201 tatgttgata aagttctaga aacaacagtg gagatattca ataagctcaa ccttgaacat 1261 attgctacca gtagtgcagt ttcaaaggaa ctcaccagac ttttgaaaat accagttgac 1321 acttacaaca atattttaac agtcttgaaa ttaaaacatt ttcacccact ctttgagtac 1381 tttgactacg agtccagaaa gagcatgagt tgttatgtgc ttagtaatgt tctggattat 1441 aacacagaaa ttgtctctca agaccaggtg gattccataa tgaatttggt atccacgttg 1501 attcaagatc agccagatca acctgtagaa gaccctgatc cagaagattt tgctgatgag 1561 cagagccttg tgggccgctt cattcatctg ctgcgctctg aggaccctga ccagcagtac 1621 ttgattttga acacagcacg aaaacatttt ggagctggtg gaaatcagcg gattcgcttc 1681 acactgccac ctttggtatt tgcagcttac cagctggctt ttcgatataa agagaattct 1741 aaagtggatg acaaatggga aaagaaatgc cagaagattt tttcatttgc ccaccagact 1801 atcagtgctt tgatcaaagc agagctggca gaattgccct taagactttt tcttcaagga 1861 gcactagctg ctggggaaat tggttttgaa aatcatgaga cagtcgcata tgaattcatg 1921 tcccaggcat tttctctgta tgaagatgaa atcagcgatt ccaaagcaca gctagctgcc 1981 atcaccttga tcattggcac ttttgaaagg atgaagtgct tcagtgaaga gaatcatgaa 2041 cctctgagga ctcagtgtgc ccttgctgca tccaaacttc taaagaaacc tgatcagggc 2101 cgagctgtga gcacctgtgc acatctcttc tggtctggca gaaacacgga caaaaatggg 2161 gaggagcttc acggaggcaa gagggtaatg gagtgcctaa aaaaagctct aaaaatagca 2221 aatcagtgca tggacccctc tctacaagtg cagcttttta tagaaattct gaacagatat 2281 atctattttt atgaaaagga aaatgatgcg gtaacaattc aggttttaaa ccagcttatc 2341 caaaagattc gagaagacct cccgaatctt gaatccagtg aagaaacaga gcagattaac 2401 aaacattttc ataacacact ggagcatttg cgcttgcggc gggaatcacc agaatccgag 2461 gggccaattt atgaaggtct catcctttaa aaaggaaata gctcaccata ctcctttcca 2521 tgtacatcca gtgagggttt tattacgcta ggtttccctt ccatagattg tgcctttcag 2581 aaatgctgag gtaggtttcc catttcttac ctgtgatgtg ttttacccag cacctccgga 2641 cactcacctt caggacctta ataaaattat tcacttggta agtgttcaag tctttctgat 2701 caccccaagt agcatgactg atctgcaatt taaaattcct gtgatctgta aaaaaaaaaa 2761 aaaaaaaaaa aacaaaaccc acaagcactt atcttggcta ctaatgaagc tctccttttt 2821 tttgtttgtt tgtttgcttc attgttgatt gtgtattttc ttcattcctg gggagtacta 2881 acccaaaagc gtctgtctct tgttttctag tccagtttga gattaattta gaagaaagga 2941 atactgtatg tgaaattcat cttgggcttt cccctaaatt gcaagataag gccatgtgta 3001 agattttccc taaaactaga atatattaat gcatgtttga gaattttaaa gcaccatggt 3061 caaaaccaga agctatattt tgcatatttg gactcagcca tccattaaga acccatgttg 3121 tcctctggac atatttatca atataattgg gttttaaata gtataaaaga aaacttgtga 3181 tctatataat ttatgtatca ccttcattgt aaatttagca ggaaatgcat cacaattatg 3241 attttttttt ttgcaccagt gaaacaataa agatgctatt aacaaaaaaa aaaaaaaa

For example, the polypeptide sequence corresponding to human VPS35 is encoded by the nucleic acid sequence of SEQ ID NO: 13 and is depicted in SEQ ID NO: 14 (796aa). Sequence information related to VPS35 is accessible in public databases by GenBank Accession numbers NP_060676.2 (protein).

  1 MPTTQQSPQD EQEKLLDEAI QAVKVQSFQM KRCLDKNKLM DALKHASNML GELRTSMLSP  61 KSYYELYMAI SDELHYLEVY LTDEFAKGRK VADLYELVQY AGNIIPRLYL LITVGVVYVK 121 SFPQSRKDIL KDLVEMCRGV QHPLRGLFLR NYLLQCTRNI LPDEGEPTDE ETTGDISDSM 181 DFVLLNFAEM NKLWVRMQHQ GHSRDREKRE RERQELRILV GTNLVRLSQL EGVNVERYKQ 241 IVLTGILEQV VNCRDALAQE YLMECIIQVF PDEFHLQTLN PFLRACAELH QNVNVKNIII 301 ALIDRLALFA HREDGPGIPA DIKLFDIFSQ QVATVIQSRQ DMPSEDVVSL QVSLINLAMK 361 CYPDRVDYVD KVLETTVEIF NKLNLEHIAT SSAVSKELTR LLKIPVDTYN NILTVLKLKH 421 FHPLFEYFDY ESRKSMSCYV LSNVLDYNTE IVSQDQVDSI MNLVSTLIQD QPDQPVEDPD 481 PEDFADEQSL VGRFIHLLRS EDPDQQYLIL NTARKHFGAG GNQRIRFTLP PLVFAAYQLA 541 FRYKENSKVD DKWEKKCQKI FSFAHQTISA LIKAELAELP LRLFLQGALA AGEIGFENHE 601 TVAYEFMSQA FSLYEDEISD SKAQLAAITL IIGTFERMKC FSEENHEPLR TQCALAASKL 661 LKKPDQGRAV STCAHLFWSG RNTDKNGEEL HGGKRVMECL KKALKIANQC MDPSLQVQLF 721 IEILNRYIYF YEKENDAVTI QVLNQLIQKI REDLPNLESS EETEQINKHF HNTLEHLRLR 781 RESPESEGPI YEGLIL

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 1) has a mutation wherein the amino acid at position 67 is a lysine (L) instead of a glutamine (Q) and is depicted in SEQ ID NO: 26 (203aa).

SEQ ID NO: 26:   1 MGSRDHLFKV LVVGDAAVGK TSLVQRYSQD SFSKHYKSTV GVDFALKVLQ WSDYEIVRLQ  61 LWDIAGLERF TSMTRLYYRD ASACVIMFDV TNATTFSNSQ RWKQDLDSKL TLPNGEPVPC 121 LLLANKCDLS PWAVSRDQID RFSKENGFTG WTETSVKENK NINEAMRVLI EKMMRNSTED 181 IMSLSTQGDY INLQTKSSSW SCC

For example, the polypeptide sequence corresponding to human LRRK2 has a mutation wherein the amino acid at position 2019 is a serine (S) instead of a glycine (G) and is depicted in SEQ ID NO: 27 (2527aa).

SEQ ID NO: 27:    1 MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSEH ASKLFQGKNI   61 HVPLLIVLDS YMRVASVQQV GWSLLCKLIE VCPGTMQSLM GPQDVGNDWE VLGVHQLILK  121 MLTVHNASVN LSVIGLKTLD LLLTSGKITL LILDEESDIF MLIFDAMHSF PANDEVQKLG  181 CKALHVLFER VSEEQLTEFV ENKDYMILLS ALTNFKDEEE IVLHVLHCLH SLAIPCNNVE  241 VLMSGNVRCY NIVVEAMKAF PMSERIQEVS CCLLHRLTLG NFFNILVLNE VHEFVVKAVQ  301 QYPENAALQI SALSCLALLT ETIFLNQDLE EKNENQENDD EGEEDKLFWL EACYKALTWH  361 RKNKHVQEAA CWALNNLLMY QNSLHEKIGD EDGHFPAHRE VMLSMLMHSS SKEVFQASAN  421 ALSTLLEQNV NFRKILLSKG IHLNVLELMQ KHIHSPEVAE SGCKMLNHLF EGSNTSLDIM  481 AAVVPKILTV MKRHETSLPV QLEALRAILH FIVPGMPEES REDTEFHHKL NMVKKQCFKN  541 DIHKLVLAAL NRFIGNPGIQ KCGLKVISSI VHFPDALEML SLEGAMDSVL HTLQMYPDDQ  601 EIQCLGLSLI GYLITKKNVF IGTGHLLAKI LVSSLYRFKD VAEIQTKGFQ TILAILKLSA  661 SFSKLLVHHS FDLVIFHQMS SNIMEQKDQQ FLNLCCKCFA KVAMDDYLKN VMLERACDQN  721 NSIMVECLLL LGADANQAKE GSSLICQVCE KESSPKLVEL LLNSGSREQD VRKALTISIG  781 KGDSQIISLL LRRLALDVAN NSICLGGFCI GKVEPSWLGP LFPDKTSNLR KQTNIASTLA  841 RMVIRYQMKS AVEEGTASGS DGNFSEDVLS KFDEWTFIPD SSMDSVFAQS DDLDSEGSEG  901 SFLVKKKSNS ISVGEFYRDA VLQRCSPNLQ RHSNSLGPIF DHEDLLKRKR KILSSDDSLR  961 SSKLQSHMRH SDSISSLASE REYITSLDLS ANELRDIDAL SQKCCISVHL EHLEKLELHQ 1021 NALTSFPQQL CETLKSLTHL DLHSNKFTSF PSYLLKMSCI ANLDVSRNDI GPSVVLDPTV 1081 KCPTLKQFNL SYNQLSFVPE NLTDVVEKLE QLILEGNKIS GICSPLRLKE LKILNLSKNH 1141 ISSLSENFLE ACPKVESFSA RMNFLAAMPF LPPSMTILKL SQNKFSCIPE AILNLPHLRS 1201 LDMSSNDIQY LPGPAHWKSL NLRELLFSHN QISILDLSEK AYLWSRVEKL HLSHNKLKEI 1261 PPEIGCLENL TSLDVSYNLE LRSFPNEMGK LSKIWDLPLD ELHLNFDFKH IGCKAKDIIR 1321 FLQQRLKKAV PYNRMKLMIV GNTGSGKTTL LQQLMKTKKS DLGMQSATVG IDVKDWPIQI 1381 RDKRKRDLVL NVWDFAGREE FYSTHPHFMT QRALYLAVYD LSKGQAEVDA MKPWLFNIKA 1441 RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA TEESDALAKL 1501 RKTIINESLN FKIRDQLVVG QLIPDCYVEL EKIILSERKN VPIEFPVIDR KRLLQLVREN 1561 QLQLDENELP HAVHFLNESG VLLHFQDPAL QLSDLYFVEP KWLCKIMAQI LTVKVEGCPK 1621 HPKGIISRRD VEKFLSKKRK FPKNYMSQYF KLLEKFQIAL PIGEEYLLVP SSLSDHRPVI 1681 ELPHCENSEI IIRLYEMPYF PMGFWSRLIN RLLEISPYML SGRERALRPN RMYWRQGIYL 1741 NWSPEAYCLV GSEVLDNHPE SFLKITVPSC RKGCILLGQV VDHIDSLMEE WFPGLLEIDI 1801 CGEGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI 1861 LADLPRNIML NNDELEFEQA PEFLLGDGSF GSVYRAAYEG EEVAVKIFNK HTSLRLLRQE 1921 LVVLCHLHHP SLISLLAAGI RPRMLVMELA SKGSLDRLLQ QDKASLTRTL QHRIALHVAD 1981 GLRYLHSAMI IYRDLKPHNV LLFTLYPNAA IIAKIADYSI AQYCCRMGIK TSEGTPGFRA 2041 PEVARGNVIY NQQADVYSFG LLLYDILTTG GRIVEGLKFP NEFDELEIQG KLPDPVKEYG 2101 CAPWPMVEKL IKQCLKENPQ ERPTSAQVFD ILNSAELVCL TRRILLPKNV IVECMVATHH 2161 NSRNASIWLG CGHTDRGQLS FLDLNTEGYT SEEVADSRIL CLALVHLPVE KESWIVSGTQ 2221 SGTLLVINTE DGKKRHTLEK MTDSVTCLYC NSFSKQSKQK NFLLVGTADG KLAIFEDKTV 2281 KLKGAAPLKI LNIGNVSTPL MCLSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT 2341 SQLFSYAAFS DSNIITVVVD TALYIAKQNS PVVEVWDKKT EKLCGLIDCV HFLREVMVKE 2401 NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCNSVRVMMT 2461 AQLGSLKNVM LVLGYNRKNT EGTQKQKEIQ SCLTVWDINL PHEVQNLEKH IEVRKELAEK 2521 MRRTSVE

For example, the polypeptide sequence corresponding to human LRRK2 has a mutation wherein the amino acid at position 1441 is a cysteine (C) instead of an arginine (R) and is depicted in SEQ ID NO: 28 (2527aa).

SEQ ID NO: 28:    1 MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSEH ASKLFQGKNI   61 HVPLLIVLDS YMRVASVQQV GWSLLCKLIE VCPGTMQSLM GPQDVGNDWE VLGVHQLILK  121 MLTVHNASVN LSVIGLKTLD LLLTSGKITL LILDEESDIF MLIFDAMHSF PANDEVQKLG  181 CKALHVLFER VSEEQLTEFV ENKDYMILLS ALTNFKDEEE IVLHVLHCLH SLAIPCNNVE  241 VLMSGNVRCY NIVVEAMKAF PMSERIQEVS CCLLHRLTLG NFFNILVLNE VHEFVVKAVQ  301 QYPENAALQI SALSCLALLT ETIFLNQDLE EKNENQENDD EGEEDKLFWL EACYKALTWH  361 RKNKHVQEAA CWALNNLLMY QNSLHEKIGD EDGHFPAHRE VMLSMLMHSS SKEVFQASAN  421 ALSTLLEQNV NFRKILLSKG IHLNVLELMQ KHIHSPEVAE SGCKMLNHLF EGSNTSLDIM  481 AAVVPKILTV MKRHETSLPV QLEALRAILH FIVPGMPEES REDTEFHHKL NMVKKQCFKN  541 DIHKLVLAAL NRFIGNPGIQ KCGLKVISSI VHFPDALEML SLEGAMDSVL HTLQMYPDDQ  601 EIQCLGLSLI GYLITKKNVF IGTGHLLAKI LVSSLYRFKD VAEIQTKGFQ TILAILKLSA  661 SFSKLLVHHS FDLVIFHQMS SNIMEQKDQQ FLNLCCKCFA KVAMDDYLKN VMLERACDQN  721 NSIMVECLLL LGADANQAKE GSSLICQVCE KESSPKLVEL LLNSGSREQD VRKALTISIG  781 KGDSQIISLL LRRLALDVAN NSICLGGFCI GKVEPSWLGP LFPDKTSNLR KQTNIASTLA  841 RMVIRYQMKS AVEEGTASGS DGNFSEDVLS KFDEWTFIPD SSMDSVFAQS DDLDSEGSEG  901 SFLVKKKSNS ISVGEFYRDA VLQRCSPNLQ RHSNSLGPIF DHEDLLKRKR KILSSDDSLR  961 SSKLQSHMRH SDSISSLASE REYITSLDLS ANELRDIDAL SQKCCISVHL EHLEKLELHQ 1021 NALTSFPQQL CETLKSLTHL DLHSNKFTSF PSYLLKMSCI ANLDVSRNDI GPSVVLDPTV 1081 KCPTLKQFNL SYNQLSFVPE NLTDVVEKLE QLILEGNKIS GICSPLRLKE LKILNLSKNH 1141 ISSLSENFLE ACPKVESFSA RMNFLAAMPF LPPSMTILKL SQNKFSCIPE AILNLPHLRS 1201 LDMSSNDIQY LPGPAHWKSL NLRELLFSHN QISILDLSEK AYLWSRVEKL HLSHNKLKEI 1261 PPEIGCLENL TSLDVSYNLE LRSFPNEMGK LSKIWDLPLD ELHLNFDFKH IGCKAKDIIR 1321 FLQQRLKKAV PYNRMKLMIV GNTGSGKTTL LQQLMKTKKS DLGMQSATVG IDVKDWPIQI 1381 RDKRKRDLVL NVWDFAGREE FYSTHPHFMT QRALYLAVYD LSKGQAEVDA MKPWLFNIKA 1441 CASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA TEESDALAKL 1501 RKTIINESLN FKIRDQLVVG QLIPDCYVEL EKIILSERKN VPIEFPVIDR KRLLQLVREN 1561 QLQLDENELP HAVHFLNESG VLLHFQDPAL QLSDLYFVEP KWLCKIMAQI LTVKVEGCPK 1621 HPKGIISRRD VEKFLSKKRK FPKNYMSQYF KLLEKFQIAL PIGEEYLLVP SSLSDHRPVI 1681 ELPHCENSEI IIRLYEMPYF PMGFWSRLIN RLLEISPYML SGRERALRPN RMYWRQGIYL 1741 NWSPEAYCLV GSEVLDNHPE SFLKITVPSC RKGCILLGQV VDHIDSLMEE WFPGLLEIDI 1801 CGEGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI 1861 LADLPRNIML NNDELEFEQA PEFLLGDGSF GSVYRAAYEG EEVAVKIFNK HTSLRLLRQE 1921 LVVLCHLHHP SLISLLAAGI RPRMLVMELA SKGSLDRLLQ QDKASLTRTL QHRIALHVAD 1981 GLRYLHSAMI IYRDLKPHNV LLFTLYPNAA IIAKIADYGI AQYCCRMGIK TSEGTPGFRA 2041 PEVARGNVIY NQQADVYSFG LLLYDILTTG GRIVEGLKFP NEFDELEIQG KLPDPVKEYG 2101 CAPWPMVEKL IKQCLKENPQ ERPTSAQVFD ILNSAELVCL TRRILLPKNV IVECMVATHH 2161 NSRNASIWLG CGHTDRGQLS FLDLNTEGYT SEEVADSRIL CLALVHLPVE KESWIVSGTQ 2221 SGTLLVINTE DGKKRHTLEK MTDSVTCLYC NSFSKQSKQK NFLLVGTADG KLAIFEDKTV 2281 KLKGAAPLKI LNIGNVSTPL MCLSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT 2341 SQLFSYAAFS DSNIITVVVD TALYIAKQNS PVVEVWDKKT EKLCGLIDCV HFLREVMVKE 2401 NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCNSVRVMMT 2461 AQLGSLKNVM LVLGYNRKNT EGTQKQKEIQ SCLTVWDINL PHEVQNLEKH IEVRKELAEK 2521 MRRTSVE

A RAB7L1, a LRRK2, or a VPS35 molecule can also encompass ortholog genes, which are genes conserved among different biological species such as humans, dogs, cats, mice, and rats, that encode proteins (for example, homologs (including splice variants), mutants, and derivatives) having biologically equivalent functions as the human-derived protein. Orthologs of a RAB7L1, a LRRK2, or a VPS35 protein include any mammalian ortholog inclusive of the ortholog in humans and other primates, experimental mammals (such as mice, rats, hamsters and guinea pigs), mammals of commercial significance (such as horses, cows, camels, pigs and sheep), and also companion mammals (such as domestic animals, e.g., rabbits, ferrets, dogs, and cats). A RAB7L1, a LRRK2, or a VPS35 molecule can comprise a protein encoded by a nucleic acid sequence homologous to the human nucleic acid, wherein the nucleic acid is found in a different species and wherein that homolog encodes a protein similar to a RAB7L1, a LRRK2, or a VPS35 protein.

The invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (2001).

One skilled in the art can obtain RAB7L1, a LRRK2, or a VPS35 molecule, in several ways, which include, but are not limited to, isolating the protein via biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods.

The invention provides for a RAB7L1, a LRRK2, or a VPS35 molecule that are encoded by nucleotide sequences. The RAB7L1, LRRK2, or VPS35 molecule can be a polypeptide encoded by a nucleic acid (including genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA). For example, a RAB7L1, a LRRK2, or a VPS35 molecule can be encoded by a recombinant nucleic acid encoding a human RAB7L1, a human LRRK2, or a human VPS35 protein, or fragment thereof. The RAB7L1, LRRK2, or VPS35 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a RAB7L1, a LRRK2, or a VPS35 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. The RAB7L1, LRRK2, or VPS35 molecule of the invention can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. A RAB7L1, a LRRK2, or a VPS35 molecule of this invention can also encompasses variants of the human RAB7L1, LRRK2, or VPS35 proteins. The variants can comprise naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms.

In one embodiment, a fragment of a nucleic acid sequence that comprises a RAB7L1, a LRRK2, or a VPS35 molecule can encompass any portion of at least about 8 consecutive nucleotides of SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In one embodiment, the fragment can comprise at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides of SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. Fragments include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100 nucleotides.

A RAB7L1, a LRRK2, or a VPS35 molecule, can be a fragment of a RAB7L1, a LRRK2, or a VPS35 protein. For example, the RAB7L1, LRRK2, or VPS35 protein fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 6, 7, 8, 11, 14, 26, 27, or 28. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, at least about 80 consecutive amino acids, at least about 90 consecutive amino acids, at least about 100 consecutive amino acids, at least about 110 consecutive amino acids, or at least about 120 consecutive amino acids of SEQ ID NOS: 6, 7, 8, 11, 14, 26, 27, or 28. Fragments include all possible amino acid lengths between about 8 and 80 about amino acids, for example, lengths between about 10 and about 80 amino acids, between about 15 and about 80 amino acids, between about 20 and about 80 amino acids, between about 35 and about 80 amino acids, between about 40 and about 80 amino acids, between about 50 and about 80 amino acids, or between about 70 and about 80 amino acids.

Methods of treating Parkinson's Disease

In one aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD, and (b) administering a treatment if the subject has an increased risk or predisposition to PD.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD, and (b) administering a treatment if the subject has an increased risk or predisposition to PD.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD, and (b) administering a treatment if the subject has an increased risk or predisposition to PD.

In another aspect, the invention provides methods for treating PD in a subject, comprising administering to a subject in need thereof a therapeutic amount of a retromer complex-stabilizing compound, or a pharmaceutically acceptable salt thereof.

In some embodiments, the retromer complex-stabilizing compound stabilizes VPS35, VPS29, VPS26 or a combination thereof. In some embodiments, the retromer complex-stabilizing compound binds to VPS35 and VPS29. In some embodiments, the retromer complex-stabilizing compound stabilizes the interaction between VPS35 and VPS29. In some embodiments, the retromer complex-stabilizing compound is R55. In other embodiments, the retromer complex-stabilizing compound is R33.

As used herein, “single-nucleotide polymorphism” or “SNP” refers to variations at single-nucleotide positions in the DNA sequence among individuals. Information on SNPs can be found in publically accessible databases, such as, in the SNP database at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). In one embodiment, the genetic variant at the PARK 16 locus comprises single-nucleotide polymorphism (SNP) rs823114, SNP rs823154, SNP rs823128, SNP rs947211, or a combination thereof.

In one embodiment, the PARK16 locus comprises the genes SLC45A3, NUCKS1, RAB7L1, SLC41A1, and PM20D1. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant at the SLC45A3, NUCKS1, SLC41A1, or PM20D1 gene. In one embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931.

Without being bound by theory, genetic variants can be associated with PD. In one embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931.

Genetic variants can also affect the splicing of mRNA. Without being bound by theory, pre-mRNA transcribed from genomic DNA can be spliced so that introns are removed and exons are joined together. Transcribed pre-mRNA can be alternatively spliced creating a range of unique proteins (known as “isoforms”) and/or mRNAs (known as “transcript variants”) by varying the exon composition of the mRNA. In one embodiment, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence.

In one embodiment, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. Various mutations that affect the transcription and translation of a RAB7L1 molecule can result in loss of expression of a RAB7L1 protein.

In one embodiment, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In another embodiment, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In another embodiment, the protein of SEQ ID NO: 27 or 28 is associated with familial PD. In another embodiment, the genetic variant at the LRRK2 locus is associated with sporadic, or non-familial PD.

In one embodiment, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. Various mutations that affect the transcription and translation of a LRRK2 molecule can result in loss of expression of a LRRK2 protein.

In one aspect the invention provides, a method of treating PD in a subject comprising: (a) measuring the expression levels of a set of genes in a sample from a subject, wherein the set of genes comprises at least one gene selected from the genes listed in Table 2 (b) comparing the expression levels of the set of genes in the subject sample to expression levels of the same set of genes in a reference sample or samples, wherein the reference sample or samples are from an individual who has a PD-associated SNP, and wherein similar expression levels of the set of genes in the subject sample and the set of genes in the reference sample(s) indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD.

In another aspect, the invention provides a method of treating PD in a subject comprising: (a) determining a level of full-length RAB7L1 in a sample from a subject, (b) comparing the level of full-length RAB7L1 from the subject sample to a full-length RAB7L1 level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the full-length RAB7L in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of full-length RAB7L is protein level of full-length RAB7L, or mRNA levels of the full-length RAB7L, or a combination thereof.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining a level of isoform 3 of RAB7L1 in a sample from a subject, (b) comparing the level of isoform 3 of RAB7L1 from the subject sample to an isoform 3 of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of isoform 3 of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of isoform 3 of RAB7L1 is a protein level. In one embodiment, the method further comprises determining the level of transcript variant 4, 5, or a combination thereof of RAB7L1.

A method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining a level of transcript variant 4, 5, or a combination thereof of RAB7L1 in a sample from a subject, (b) comparing the level of transcript variant 4, 5, or a combination thereof of RAB7L1 from the subject sample to a transcript variant 4, 5, or a combination thereof of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of transcript variant 4, 5, or a combination thereof of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of transcript variant 4, 5, or a combination thereof of RAB7L1 is a mRNA level. In another embodiment, the method further comprises determining the level of isoform 3 of RAB7L1.

In one embodiment, the invention provides for determine the level of retromer components. Without being bound by theory, retromer is a complex of proteins which are involved in recycling between the endolysosomal compartment of a cell and the Golgi apparatus. In mammals, proteins of the retromer complex include, but are not limited to Vps26, Vps29, Vps35, SNX1, SNX2, SNX5 and SNX6. The retromer complex can act in two subcomplexes; a cargo recognition complex that comprises Vps35, Vps29 and Vps26 (Vps trimer), and SNX-BAR dimers that comprises SNX1 and SNX2 or SNX5 and SNX6.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining a level of retromer components in a sample from a subject, (b) comparing the level of retromer components from the subject sample to a retromer component level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the retromer components in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of retromer component is protein level of retromer component, or mRNA levels of retromer component, or a combination thereof. In another embodiment, the retromer component is VPS35, VPS29, VPS26 or a combination thereof. In a further embodiment the retromer component is SNX1, SNX2, SNX5, SNX6, or a combination thereof. In one embodiment, the level of VPS35, VPS29, or VPS26 is protein level of VPS35, VPS29, or VPS26, or mRNA levels of VPS35, VPS29, or VPS26, or a combination thereof.

In one aspect, the invention provides a method of treating PD in a subject. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the protein comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, or SEQ ID NO: 14.

In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the protein comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 6, SEQ ID NO: 26, or SEQ ID NO: 14.

In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the protein comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, or SEQ ID NO: 14.

In one embodiment, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. Suitable methods for determining the PD disease status are known to one of skill in the art.

In one embodiment, the subject is not diagnosed with PD. In another embodiment, the subject is diagnosed with PD. In another embodiment, the subject is diagnosed with a pre-disease prodromal state.

In one embodiment, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. Methods and types of physical examinations are known to one of skill in the art.

In one embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

In one embodiment, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. Determination of parkinsonism symptoms are known to one of skill in the art.

In one embodiment, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. Methods of sample collection are known to one of skill in the art.

Expression Systems and Purification of Recombinant Proteins

One skilled in the art understands that polypeptides (for example RAB7L1, LRRK2, or VPS35, and the like) can be obtained in several ways, which include but are not limited to, expressing a nucleotide sequence encoding the protein of interest, or fragment thereof, by genetic engineering methods.

In one embodiment, the nucleic acid is expressed in an expression cassette, for example, to achieve overexpression in a cell. The nucleic acids of the invention can be an RNA, cDNA, cDNA-like, or a DNA of interest in an expressible format, such as an expression cassette, which can be expressed from the natural promoter or an entirely heterologous promoter. The nucleic acid of interest can encode a protein, and may or may not include introns. Any recombinant expression system can be used, including, but not limited to, bacterial, mammalian, yeast, insect, or plant cell expression systems.

Host cells transformed with a nucleic acid sequence encoding a RAB7L1, a LRRK2, or a VPS35 molecule can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. Expression vectors containing a nucleic acid sequence encoding a RAB7L1, a LRRK2, or a VPS35 molecule can be designed to contain signal sequences which direct secretion of soluble polypeptide molecules encoded by a RAB7L1, a LRRK2, or a VPS35 molecule, through a prokaryotic or eukaryotic cell membrane.

Nucleic acid sequences comprising a RAB7L1, a LRRK2, or a VPS35 molecule that encode a polypeptide can be synthesized, in whole or in part, using chemical methods known in the art. Alternatively, a RAB7L1, a LRRK2, or a VPS35 molecule can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of a RAB7L1, a LRRK2, or a VPS35 molecule can be separately synthesized and combined using chemical methods to produce a full-length molecule.

A synthetic peptide can be substantially purified via high performance liquid chromatography (HPLC). The composition of a synthetic RAB7L1, LRRK2, or VPS35 molecule can be confirmed by amino acid analysis or sequencing. Additionally, any portion of an amino acid sequence comprising a protein encoded by a RAB7L1, a LRRK2, or a VPS35 molecule can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

The invention further encompasses methods for using a protein or polypeptide encoded by a nucleic acid sequence of a RAB7L1, a LRRK2, or a VPS35 molecule, such as the sequences shown in SEQ ID NOS: 6, 7, 8, 11, 14, 26, 27, or 28. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a RAB7L1, a LRRK2, or a VPS35 molecule has the amino acid sequence shown in either SEQ ID NO: 6, 7, 8, 11, 14, 26, 27, or 28. In certain embodiments, the invention encompasses variants of a human protein encoded by a RAB7L1, a LRRK2, or a VPS35 molecule.

One skilled in the art understands that expression of desired protein products can be carried out in prokaryotes (e.g. E. coli and B. subtilis), in plant cell systems infected with recombinant virus expression vectors (e.g., tobacco mosaic virus, TMV; cauliflower mosaic virus, CaMV), in insect cells (e.g. Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae), in yeast cells (e.g. Saccharomyces sp., Pichia sp.), or in mammalian cells (e.g. BHK cells, VERO cells, CHO cells and the like).

Expression vectors (also known in the art as fusion-vectors) can be designed to add a number of amino acid residues, usually to the N-terminus of the expressed recombinant protein. Such fusion vectors can serve three functions: 1) to increase the solubility of the desired recombinant protein; 2) to increase expression of the recombinant protein of interest; and 3) to aid in recombinant protein purification by acting as a ligand in affinity purification.

An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection, or electroporation. Electroporation is carried out at approximate voltage and capacitance to result in entry of the DNA construct(s) into cells of interest. Other methods used to transfect cells can also include modified calcium phosphate precipitation, polybrene precipitation, liposome fusion, and receptor-mediated gene delivery.

Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland W L, et al., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cell culturing conditions can vary according to the type of host cell selected. Commercially available medium can be utilized.

A RAB7L1, a LRRK2, or a VPS35 molecule can be purified from any human or non-human cell which expresses the polypeptide, including those which have been transfected with expression constructs that express a RAB7L1, a LRRK2, or a VPS35 molecule. A purified RAB7L1, LRRK2, or VPS35 molecule can be separated from other compounds which normally associate with the RAB7L1, LRRK2, or VPS35 molecules, in the cell, such as certain proteins, carbohydrates, or lipids, using methods practiced in the art. The desired polypeptide molecule (for example, a RAB7L1, a LRRK2, or a VPS35 molecule) is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques. Non-limiting purification methods for proteins include: size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.

Methods of Administration

Nucleic Acid Delivery Methods.

The invention provides methods for treating Parkinson's Disease (PD) in a subject. In one embodiment, the method can comprise administering to the subject a RAB7L1, a LRRK2, or a VPS35 molecule (e.g., a RAB7L1, a LRRK2, or a VPS35 polypeptide or a RAB7L1, a LRRK2, or a VPS35 polynucleotide).

Various approaches can be carried out to restore the activity or function of a RAB7L1, a LRRK2, or a VPS35 molecule in a subject, such as those carrying an genetic variant in a RAB7L1, a LRRK2, or a VPS35 gene locus. For example, supplying wild-type RAB7L1, LRRK2, or VPS35 gene function to such subjects can treat Parkinson's Disease. Increasing a RAB7L1, a LRRK2, or a VPS35 gene expression level or activity can be accomplished through gene or protein therapy.

A nucleic acid encoding a RAB7L1, a LRRK2, or a VPS35 molecule can be introduced into the cells of a subject. For example, the wild-type gene (or fragment thereof) can also be introduced into the cells of the subject in need thereof using a vector as described herein. The vector can be a viral vector or a plasmid. The gene can also be introduced as naked DNA. The gene can be provided so as to integrate into the genome of the recipient host cells, or to remain extra-chromosomal. Integration can occur randomly or at precisely defined sites, such as through homologous recombination. For example, a functional copy of a RAB7L1, a LRRK2, or a VPS35 molecule can be inserted in replacement of an altered version in a cell, through homologous recombination. Further techniques include gene gun, liposome-mediated transfection, or cationic lipid-mediated transfection. Gene therapy can be accomplished by direct gene injection, or by administering ex vivo prepared genetically modified cells expressing a functional polypeptide.

Delivery of nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (see, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)). Introduction of a nucleic acid or a gene encoding a polypeptide of the invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of therapeutic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapy vectors. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., (1992) J Gen Virol. 73(Pt 6):1533-6), adenovirus (Berkner (1992) Curr Top Microbiol Immunol. 158:39-66; Berkner (1988) Biotechniques, 6(7):616-29; Gorziglia and Kapikian (1992) J Virol. 66(7):4407-12; Quantin et al., (1992) Proc Natl Acad Sci USA. 89(7):2581-4; Rosenfeld et al., (1992) Cell. 68(1):143-55; Wilkinson et al., (1992) Nucleic Acids Res. 20(9):2233-9; Stratford-Perricaudet et al., (1990) Hum Gene Ther. 1(3):241-56), vaccinia virus (Moss (1992) Curr Opin Biotechnol. 3(5):518-22), adeno-associated virus (Muzyczka, (1992) Curr Top Microbiol Immunol. 158:97-129; Ohi et al., (1990) Gene. 89(2):279-82), herpesviruses including HSV and EBV (Margolskee (1992) Curr Top Microbiol Immunol. 158:67-95; Johnson et al., (1992) Brain Res Mol Brain Res. 12(1-3):95-102; Fink et al., (1992) Hum Gene Ther. 3(1):11-9; Breakefield and Geller (1987) Mol Neurobiol. 1(4):339-71; Freese et al., (1990) Biochem Pharmacol. 40(10):2189-99), and retroviruses of avian (Bandyopadhyay and Temin (1984) Mol Cell Biol. 4(4):749-54; Petropoulos et al., (1992) J Virol. 66(6):3391-7), murine (Miller et al. (1992) Mol Cell Biol. 12(7):3262-72; Miller et al., (1985) J Virol. 55(3):521-6; Sorge et al., (1984) Mol Cell Biol. 4(9):1730-7; Mann and Baltimore (1985) J Virol. 54(2):401-7; Miller et al., (1988) J Virol. 62(11):4337-45), and human origin (Shimada et al., (1991) J Clin Invest. 88(3):1043-7; Helseth et al., (1990) J Virol. 64(12):6314-8; Page et al., (1990) J Virol. 64(11):5270-6; Buchschacher and Panganiban (1992) J Virol. 66(5):2731-9).

Non-limiting examples of in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993), incorporated entirely by reference). For example, naked DNA vaccines are generally known in the art; see Brower, Nature Biotechnology, 16:1304-1305 (1998), which is incorporated by reference in its entirety. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

For reviews of gene therapy protocols and methods see Anderson et al., Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847; 8,398,968; and 8,404,653 which are all hereby incorporated by reference in their entireties. For an example of gene therapy treatment in humans see Porter et al., NEJM 2011 365:725-733 and Kalos et al. Sci. Transl. Med. 2011. 201 3(95):95. For additional reviews of gene therapy technology, see Friedmann, Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990); Miller, Nature, 357: 455-460 (1992); Kikuchi et al., J Dermatol Sci. 2008 May; 50(2):87-98; Isaka et al., Expert Opin Drug Deliv. 2007 September; 4(5):561-71; Jager et al., Curr Gene Ther. 2007 August; 7(4):272-83; Waehler et al., Nat Rev Genet. 2007 August; 8(8):573-87; Jensen et al., Ann Med. 2007; 39(2):108-15; Herweijer et al., Gene Ther. 2007 January; 14(2):99-107; Eliyahu et al., Molecules, 2005 Jan. 31; 10(1):34-64; and Altaras et al., Adv Biochem Eng Biotechnol. 2005; 99:193-260, all of which are hereby incorporated by reference in their entireties.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

Protein Delivery Methods.

Protein replacement therapy can increase the amount of protein by exogenously introducing wild-type or biologically functional protein by way of infusion. A replacement polypeptide can be synthesized according to known chemical techniques or may be produced and purified via known molecular biological techniques. Protein replacement therapy has been developed for various disorders. For example, a wild-type protein can be purified from a recombinant cellular expression system (e.g., mammalian cells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No. 6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et al.; U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No. 6,451,600 to Rasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen et al. and U.S. Pat. No. 5,879,680 to Ginns et al.), human placenta, or animal milk (see U.S. Pat. No. 6,188,045 to Reuser et al.), or other sources known in the art. After the infusion, the exogenous protein can be taken up by tissues through non-specific or receptor-mediated mechanism.

A RAB7L1, a LRRK2, or a VPS35 molecule can also be delivered in a controlled release system. For example, the RAB7L1, LRRK2, or VPS35 molecule can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see Sefton (1987) Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science (1990) 249:1527-1533).

Retromer Complex-Stabilizing Compounds

In one embodiment, the invention provides for a method of treating Parkinson's Disease, comprising administering to a subject in need thereof a therapeutic amount of a retromer complex-stabilizing compound, or a pharmaceutically acceptable salt thereof. Without being bound by theory, retromer is a complex of proteins which are involved in recycling between the endolysosomal compartment of a cell and the Golgi apparatus. In mammals, proteins of the retromer complex include, but are not limited to VPS26, VPS29, VPS35, SNX1, SNX2, SNX5 and SNX6. The retromer complex can act in two subcomplexes; a cargo recognition complex that comprises Vps35, Vps29 and Vps26 (VPS trimer), and SNX-BAR dimers that comprises SNX1 and SNX2 or SNX5 and SNX6.

In some embodiments, the retromer complex-stabilizing compound is a compound of formula (I),

wherein Ar is a 5- or 6-membered aromatic ring or a 5- or 6-membered heteroaromatic ring having 1-4 heteroatoms independently selected from sulfur, nitrogen, or oxygen; X is

or H; R¹ is C₁-C₆-alkyl; A is —S—R², —S(O)R², —SO₂R², —SO₃R²,

and each R² is independently selected from H, C₁-C₆-alkyl, or phenyl.

In some embodiments, the compound of formula (I) binds to VPS35 and VPS29.

In one embodiment, the retromer complex-stabilizing compound is R55, wherein the structure of R55 is

In some embodiments, the retromer complex-stabilizing compound is R33, wherein the structure of R33 is

In some embodiments, the invention provides for a method of treating Parkinson's Disease, comprising administering to a subject in need thereof a pharmaceutical composition that comprises a retromer complex-stabilizing compound. In some embodiments, the pharmaceutical composition comprises a combination of agents that stabilize the retromer complex. Agents that stabilize the retromer complex include retromer-stabilizing compounds (e.g., R55, R33), as well as, nucleic acids and proteins encoding components of the retromer complex (e.g., VPS26, VPS29, VPS35, SNX1, SNX2, SNX5 and SNX6).

In some embodiments, a retromer complex-stabilizing compound can have the effect of stabilizing retromer complex. Retromer stability can be affected by an altered rate of association or disassociation between retromer components. In some embodiments, a retromer complex-stabilizing compound affects the interface between the components of the retromer complex, including, but not limited to VPS26, VPS29, VPS35, SNX1, SNX2, SNX5 and SNX6. In some embodiments, a retromer complex-stabilizing compound affects the interface of the VPS35-VPS29 complex such that association between the two polypeptides is altered. In some embodiments, a retromer complex-stabilizing compound affects the interface of the VPS26-VPS29-VPS35 trimer such that association between the three polypeptides is altered. In some embodiments, R55 binds and stabilizes the retromer complex at the interface of the VPS35-VPS29 complex. In some embodiments, stability of the retromer complex can be determined by measuring thermal stability of the retromer complex. Binding of R55 to the retromer complex is described in Mecozzi et al., Nat. Chem. Biol., 10:443-450 (2014), the contents of which is hereby incorporated by reference in its entirety.

Pharmaceutical Compositions and Methods of Administration

In some embodiments, a molecule or composition of the invention can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. Choice of the excipient and any accompanying elements of the composition comprising a RAB7L1, a LRRK2, or a VPS35 molecule or a retromer-stabilizing compound, such as R55, will be adapted in accordance with the route and device used for administration. In some embodiments, a composition comprising a RAB7L1, a LRRK2, or a VPS35 molecule or a retromer-stabilizing compound, such as R55, can also comprise, or be accompanied with, one or more other ingredients that facilitate the delivery or functional mobilization of the RAB7L1, LRRK2, or VPS35 molecule or retromer-stabilizing compound, such as R55.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

A RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, can be administered to the subject one time (e.g., as a single injection or deposition). Alternatively, a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, can be administered once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. Furthermore, a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, can be co-administrated with another therapeutic.

In one embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, can be co-administrated with a Parkinson's Disease drug. Some non-limiting examples of conventional PD drugs include: levodopa, carbidopa/levodopa (co-careldopa), benserazide/levodopadopamine (co-beneldopa), dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, and lisuride), MAO-B inhibitors (e.g. selegiline, and rasagiline), amantadine, and anticholinergics.

A RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, may also be used in combination with surgical or other interventional treatment regimens used for the treatment of PD.

A RAB7L1, a LRRK2, or a VPS35 molecule can be administered to a subject by any means suitable for delivering the protein, nucleic acid or compound to cells of the subject. For example, it can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.

The compositions of this invention can be formulated and administered to reduce the symptoms associated with PD by any means that produce contact of the active ingredient with the agent's site of action in the body of a human or non-human subject. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20^(th) ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers, such as PBS, Hank's solution, or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.

Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the RAB7L1, LRRK2, or VPS35 molecule, or a retromer-stabilizing compound, such as R55, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as known in the art

A composition of the invention can be administered to a subject in need thereof. Subjects in need thereof can include but are not limited to, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

A composition of the invention can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations can be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The pharmaceutical compositions of the invention (e.g., that have a therapeutic effect) can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel-caps, caplets, or powders, that are adapted for sustained release are encompassed by the invention.

In the methods described herein, a RAB7L1, a LRRK2, or a VPS35 molecule, can be administered to the subject either as RNA, in conjunction with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences which express the gene product. Suitable delivery reagents for administration of the a RAB7L1, a LRRK2, or a VPS35 molecule, include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.

The dosage administered can be a therapeutically effective amount of the composition sufficient to result in treatment of PD, and can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.

In some embodiments, the effective amount of the administered RAB7L1, LRRK2, or VPS35 molecule, or a retromer-stabilizing compound, such as R55, is at least about 0.01 μg/kg body weight, at least about 0.025 μg/kg body weight, at least about 0.05 μg/kg body weight, at least about 0.075 μg/kg body weight, at least about 0.1 μg/kg body weight, at least about 0.25 μg/kg body weight, at least about 0.5 μg/kg body weight, at least about 0.75 μg/kg body weight, at least about 1 μg/kg body weight, at least about 5 μg/kg body weight, at least about 10 μg/kg body weight, at least about 25 μg/kg body weight, at least about 50 μg/kg body weight, at least about 75 μg/kg body weight, at least about 100 μg/kg body weight, at least about 150 μg/kg body weight, at least about 200 μg/kg body weight, at least about 250 μg/kg body weight, at least about 300 μg/kg body weight, at least about 350 μg/kg body weight, at least about 400 μg/kg body weight, at least about 450 μg/kg body weight, at least about 500 μg/kg body weight, at least about 550 μg/kg body weight, at least about 600 μg/kg body weight, at least about 650 μg/kg body weight, at least about 700 μg/kg body weight, at least about 750 μg/kg body weight, at least about 800 μg/kg body weight, at least about 850 μg/kg body weight, at least about 900 μg/kg body weight, at least about 950 μg/kg body weight, at least about 1000 μg/kg body weight, at least about 1500 μg/kg body weight, at least about 2000 μg/kg body weight, at least about 2500 μg/kg body weight, at least about 3000 μg/kg body weight, at least about 3500 μg/kg body weight, at least about 4000 μg/kg body weight, at least about 4500 μg/kg body weight, at least about 5000 μg/kg body weight, at least about 5500 μg/kg body weight, at least about 6000 μg/kg body weight, at least about 6500 μg/kg body weight, at least about 7000 μg/kg body weight, at least about 7500 μg/kg body weight, at least about 8000 μg/kg body weight, at least about 8500 μg/kg body weight, at least about 9000 μg/kg body weight, at least about 9500 μg/kg body weight, or at least about 10000 μg/kg body weight.

In one embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, is administered at least once daily. In another embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, is administered at least twice daily. In some embodiments, a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, is administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 5 weeks, for at least 6 weeks, for at least 8 weeks, for at least 10 weeks, for at least 12 weeks, for at least 18 weeks, for at least 24 weeks, for at least 36 weeks, for at least 48 weeks, or for at least 60 weeks. In further embodiments, a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, is administered in combination with a second therapeutic agent.

Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Therapeutic agents that exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.

Administration of a RAB7L1, a LRRK2, or a VPS35 molecule, or a retromer-stabilizing compound, such as R55, is not restricted to a single route, but may encompass administration by multiple routes. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to one of skill in the art.

Methods of Detection

Embodiments of the invention provide for detecting expression of a RAB7L1, a LRRK2, or a VPS35 molecule. In one embodiment, a gene alteration can result in increased or reduced protein expression and/or activity. The alteration can be determined at the level of the DNA, RNA, or polypeptide.

In some embodiments, the detecting comprises detecting in a biological sample whether there is a reduction in an mRNA encoding a RAB7L1, a LRRK2, or a VPS35 protein, or a reduction in a RAB7L1, a LRRK2, or a VPS35 protein, or a combination thereof. In further embodiments, the detecting comprises detecting in a biological sample whether there is a reduction in an mRNA encoding a RAB7L1, a LRRK2, or a VPS35 protein, or a reduction in a RAB7L1, a LRRK2, or a VPS35 protein, or a combination thereof. The presence of such an alteration is indicative of the presence or predisposition to PD.

Methods for detecting and quantifying RAB7L1, LRRK2, or VPS35 molecules in biological samples are known the art. For example, protocols for detecting and measuring the expression of a polypeptide encoded by a RAB7L1, a LRRK2, or a VPS35 molecule, using either polyclonal or monoclonal antibodies specific for the polypeptide are well established. Non-limiting examples include Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

In one embodiment, a biological sample comprises, a blood sample, serum, cells (including whole cells, cell fractions, cell extracts, and cultured cells or cell lines), tissues (including tissues obtained by biopsy), body fluids (e.g., urine, sputum, amniotic fluid, synovial fluid), or from media (from cultured cells or cell lines). In one embodiment, the sample is a CSF sample, a blood sample, a plasma sample, a serum sample, or any combination thereof. The methods of detecting or quantifying RAB7L1, LRRK2, or VPS35 molecules include, but are not limited to, amplification-based assays with (signal amplification) hybridization based assays and combination amplification-hybridization assays.

Any suitable biological sample can be used in the instant methods. The biological sample can be taken from body fluid, such as urine, saliva, bone marrow, blood, and derivative blood products (sera, plasma, PBMC, circulating cells, circulating RNA). The biological sample can be taken from a human subject, from an animal, or from a cell culture. The biological sample can be obtained in vivo, in vitro or ex vivo. Non-limiting examples of biological samples include blood, serum, plasma, cerebrospinal fluid, mucus, tissue, cells, and the like, or any combination thereof. In a non-limiting embodiment the biological sample is blood. In a non-limiting embodiment the biological sample is serum. In a non-limiting embodiment the biological sample is plasma. Any suitable method to isolate nucleic acids from biological samples are contemplated for use in the invention. Biological samples for analysis are stored under suitable conditions. In non-limiting examples biological samples are kept at about 4° C. In non-limiting examples biological samples are kept at about −20° C. In non-limiting examples biological samples are kept at about −70-80° C.

A RAB7L1, a LRRK2, or a VPS35 molecule can be determined at the nucleic acid level. Optionally, detection can be determined by performing an oligonucleotide ligation assay, a confirmation based assay, a hybridization assay, a sequencing assay, an allele-specific amplification assay, a microsequencing assay, a melting curve analysis, a denaturing high performance liquid chromatography (DHPLC) assay (for example, see Jones et al, (2000) Hum Genet., 106(6):663-8), or a combination thereof. In one embodiment, the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In one embodiment, the detection is performed by sequencing all or part of a RAB7L1, a LRRK2, or a VPS35 molecule, or by selective hybridization or amplification of all or part of the RAB7L1, LRRK2, or VPS35 molecule. A nucleic acid specific amplification can be carried out before the quantification step. In one embodiment, the detecting comprises using a northern blot; real time PCR and primers directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13; a ribonuclease protection assay; a hybridization, amplification, or sequencing technique to detect a RAB7L1, a LRRK2, or a VPS35 molecule; or a combination thereof. In another embodiment, the PCR primers comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, 13, 15, 16, 17, 18, 19, 24, 25, or a combination of the primers.

Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid. A detection technique involves the use of a nucleic acid probe specific for the presence of a RAB7L1, a LRRK2, or a VPS35 molecule, followed by the detection of the presence of a hybrid. The probe can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies). The probe can be labeled to facilitate detection of hybrids. In one embodiment, the probe according to the invention can comprise a nucleic acid directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In another embodiment, the probe that detects the presence of a RAB7L1, a LRRK2, or a VPS35 molecule comprises SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

A guide to the hybridization of nucleic acids is found in e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (3^(rd) Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, 1989; Current Protocols In Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York, 2001; Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.

Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on a RAB7L1, a LRRK2, or a VPS35 molecule. In another embodiment, the sequencing can be performed using SEQ ID NO: 24, or 25.

Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction. Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele-specific PCR, or PCR based single-strand conformational polymorphism (SSCP). Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. In one embodiment, amplification comprises using forward and reverse PCR primers directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In certain subjects, the downregulation of a RAB7L1, a LRRK2, or a VPS35 molecule corresponds to a subject with PD. In one embodiment, amplification can comprise using forward and reverse PCR primers comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

Non-limiting amplification methods include, e.g., polymerase chain reaction, PCR (PCR Protocols, A Guide To Methods And Applications, ed. Innis, Academic Press, N.Y., 1990 and PCR Strategies, 1995, ed. Innis, Academic Press, Inc., N.Y.); ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh (1989) PNAS 86:1173); and, self-sustained sequence replication (Guatelli (1990) PNAS 87:1874); Q Beta replicase amplification (Smith (1997) J Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplification assay (Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario; see also Berger (1987) Methods Enzymol. 152:307-316; U.S. Pat. Nos. 4,683,195 and 4,683,202; and Sooknanan (1995) Biotechnology 13:563-564). All the references stated above are incorporated by reference in their entireties.

The invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of a RAB7L1, a LRRK2, or a VPS35 molecule. Primers can be specific for a RAB7L1, a LRRK2, or a VPS35 molecule. By using such primers, the detection of an amplification product indicates the presence of a RAB7L1, a LRRK2, or a VPS35 molecule. Examples of primers of this invention can be single-stranded nucleic acid molecules of about 8 to about 15 nucleotides in length. Perfect complementarity is useful to ensure high specificity; however, certain mismatch can be tolerated. For example, a nucleic acid primer or a pair of nucleic acid primers as described above can be used in a method for detecting the presence of a genetic variant in a subject. In one embodiment, primers can be used to detect the absence of reduced level of a RAB7L1, a LRRK2, or a VPS35 molecule. In some embodiments, the primers are directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In another embodiment, the PCR primers comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

Compositions and Kits of the Invention

In one aspect, the invention provides a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of at least two different markers in a subject sample, wherein the markers comprise LRRK2, RAB7L1 and VPS35.

In another aspect, the invention provides a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of a different marker in a subject sample, each marker being one of the genes listed in Table 2.

In one embodiment, the composition comprises a microarray, a microfluidics card, a chip, or a chamber. In another aspect, the invention provides a diagnostic kit comprising the microarray, microfluidics card, chip, or chamber.

In another aspect, the invention provides a diagnostic kit for determining the levels of RAB7L1, LRRK2, VPS35, or a combination thereof, the kit comprising at least one oligonucleotide or polynucleotide to selectively quantify the levels of RAB7L1, LRRK2, VPS35, or a combination thereof. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 15, 16, 17, or 18. In another embodiment, the oligonucleotide or polynucleotide comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 15, 16, 17, or 18.

In another aspect, the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence or absence of a PD-associated genetic variant, the kit comprising at least one oligonucleotide or polynucleotide for sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 24, or 25. In another embodiment, the oligonucleotide or polynucleotide comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 24, or 25.

The kits of the invention may also include reagents necessary or useful for the amplification of target nucleic acids, which may include, but is not limited to, DNA polymerase enzymes, primer extension deoxynucleotide triphosphates, and any buffer or other solutions generally used in PCR amplification reactions and kits.

In one embodiment, the kit can further comprise reagents and/or protocols for performing a hybridization, or amplification. In one embodiment, the kit can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from a RAB7L1, a LRRK2, or a VPS35 molecule comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NOS: 15, 16, 17, 18, 19, 24, or 25, or a combination of the primers. In one embodiment, primers can be used to detect the absence or reduction of a RAB7L1, a LRRK2, or a VPS35 molecule, such as a primer directed to SEQ ID NOS: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In another embodiment, the PCR primer comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NOS: 15, 16, 17, 18, 19, 24, or 25. In some embodiments, the kit comprises a probe for detecting a RAB7L1, a LRRK2, or a VPS35 molecule.

The diagnosis methods can be performed in vitro, ex vivo, or in vivo. These methods utilize a sample from the subject in order to assess the status of a RAB7L1, a LRRK2, or a VPS35 molecule. The sample can be any biological sample derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include, but are not limited to, fluids, tissues, cell samples, organs, and tissue biopsies. Non-limiting examples of samples include blood, liver, plasma, serum, saliva, urine, or seminal fluid. The sample can be collected according to conventional techniques and used directly for diagnosis or stored. The sample can be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments include, for instance, lysis (e.g., mechanical, physical, or chemical), centrifugation. The nucleic acids and/or polypeptides can be pre-purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides can also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. In one embodiment, the sample is contacted with reagents, such as probes or primers, in order to assess the absence or presence of a RAB7L1, a LRRK2, or a VPS35 molecule. Contacting can be performed in any suitable device, such as a plate, tube, well, or glass. In some embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate can be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, or polymers. The substrate can be of various forms and sizes, such as a slide, a membrane, a bead, a column, or a gel. The contacting can be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.

EXAMPLES

A number of Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate the exemplary modes of making and practicing the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

Example 1 LRRK2 and PARK16 PD Risk Variants Impart a Common Brain Transcriptome Impact

An unbiased and systematic approach was sought to assess the phenotypic impacts of common genetic variants associated with PD risk, particularly in brain tissue from yet unaffected carriers (FIG. 1A), in order to circumvent the limitations of the analysis of diseased patient autopsy tissue. To this end, the transcriptome-wide gene expression profiles of brain tissue samples from cohorts of unaffected individuals who share either a risk or a protective allele at any given PD risk SNP were compared (FIG. 1B). Such a Global Phenotypic Impact (GPI) quantifies the effect of disease risk variants onto the transcriptome-wide gene expression profile in brain. A key aspect of the GPI analysis herein is that tissue from unaffected individuals was tested, in hope of avoiding secondary effects of disease pathology such as cell loss.

The transcriptome-wide GPI at 7 PD-associated loci was assessed (SNCA, LRRK2, MAPT, HLA-DRA, PARK16, LAMPS, STK39, Table 1) (Simon-Sanchez et al., 2009) in a publically available gene expression dataset from cerebral cortex autopsy brain tissue of 185 individuals not apparently affected by a neurodegenerative disease (GSE15222).

TABLE 1 SNPs used for the GPI analysis and linkage with PD associated SNPs identified by GWAS. PD- Dis- associated tance Locus GPI SNPs SNPs (bp) D′ R Source LRRK2 rs7306944 rs2708453 42545 0.936 1 PDGene rs7304279 29835 0.878 0.937 PDGene MAPT rs17563787 rs1981997 243660 1 1 PDGene rs393152 94097 1 1 PDGene PARK16 rs823128 rs1620334 15533 1 1 PDGene rs823123 11966 1 1 PDGene SNCA rs356168 rs356168 0 1 1 PDGene STK39 rs10176669 rs10208207 9887 0.824 0.936 PDGene rs2102808 32166 0.365 0.869 PDGene LAMP3 rs9822789 rs11711441 18401 0.948 1 PDGene HLA-DR rs2076530 rs3129882 45714 0.484 0.67 T. H. Hamza et al.

The GPIs of the 7 loci revealed a high degree of overlap in terms of the identity of transcripts altered in expression level and the valence of such alterations: genes were coordinately altered in their expression by each of the 7 PD-associated loci (over 15-fold greater than expected by chance; p=1.5E-5 by resampling statistics; FIGS. 8A-8B, Table 2).

TABLE 2 Individual gene transcripts commonly impacted by the high-risk allele at 7 PD loci. Presented are the gene transcript-level GPI sub-components: the list of genes whose expression levels correlate with the PD high-risk allele, and in the same direction for each of the 7 PD loci studied (“SNCA”, “LRRK2”, “MAPT”, “PARK16”, “HLA-DRA”, “STK39”, “LAMP3”). Genes are identified by their Illumina probesets (“Probe”) and their Gene Symbol. Positive values correspond with a relative increase in gene expression level in the presence of a high-risk allele, negative values with a decrease. The average correlation across the 7 loci is indicated (“Average”) for each gene. Probe Gene Symbol SNCA LRRK2 MAPT PARK16 HLA-DRA STK39 LAMP3 Avg. GI_16945968-S LRP15 0.10 0.17 0.09 0.17 0.00 0.16 0.14 0.12 GI_42657118-S LOC389203 0.04 0.17 0.13 0.09 0.07 0.17 0.11 0.11 GI_30089996-A BAF53A 0.01 0.12 0.17 0.11 0.08 0.07 0.21 0.11 GI_39777591-S SLC2A10 0.09 0.25 0.11 0.01 0.10 0.16 0.03 0.11 GI_16554595-A IER3 0.03 0.14 0.10 0.11 0.20 0.02 0.14 0.11 GI_38788371-S AQR 0.11 0.13 0.15 0.06 0.04 0.02 0.22 0.10 GI_23238230-A HMGN3 0.04 0.12 0.18 0.07 0.03 0.10 0.20 0.10 GI_7705400-S HDCMA18P 0.05 0.17 0.21 0.06 0.08 0.06 0.12 0.10 GI_4503932-S GATM 0.04 0.15 0.19 0.03 0.02 0.11 0.19 0.10 GI_21450827-S MGC7036 0.01 0.10 0.13 0.13 0.08 0.16 0.10 0.10 GI_22748758-S MGC40157 0.04 0.08 0.13 0.06 0.04 0.15 0.20 0.10 GI_13376994-S ME2 0.05 0.12 0.18 0.05 0.02 0.10 0.18 0.10 GI_18860915-S XRN2 0.08 0.13 0.14 0.00 0.07 0.08 0.20 0.10 GI_8922630-S C14orf114 0.07 0.22 0.17 0.03 0.01 0.10 0.10 0.10 GI_34335150-S RPS15A 0.10 0.06 0.13 0.05 0.08 0.07 0.22 0.10 GI_38502322-S C9orf10OS 0.04 0.17 0.16 0.05 0.00 0.05 0.22 0.10 GI_42734361-S DOCK7 0.10 0.10 0.18 0.05 0.01 0.18 0.06 0.10 GI_6006027-S NRAS 0.05 0.19 0.12 0.04 0.13 0.07 0.08 0.10 GI_4507668-S TPT1 0.02 0.06 0.08 0.11 0.08 0.09 0.24 0.10 GI_31543202-S MGC8974 0.03 0.15 0.22 0.07 0.06 0.08 0.06 0.10 GI_41393567-S HEBP2 0.02 0.25 0.09 0.06 0.05 0.11 0.08 0.09 GI_37551941-S LOC284347 0.09 0.13 0.08 0.04 0.03 0.14 0.13 0.09 GI_28872862-S KIAA1194 0.10 0.08 0.12 0.05 0.13 0.04 0.12 0.09 GI_4809273-S ANXA5 0.03 0.11 0.03 0.10 0.16 0.06 0.15 0.09 GI_23065549-S GSTM2 0.02 0.07 0.17 0.10 0.07 0.11 0.11 0.09 GI_4507894-S VIM 0.01 0.17 0.07 0.08 0.04 0.10 0.16 0.09 GI_4557730-S LTBP1 0.03 0.08 0.10 0.08 0.17 0.08 0.09 0.09 GI_34222292-S GYS1 0.01 0.10 0.10 0.17 0.14 0.07 0.05 0.09 GI_37540659-S KIAA1345 0.15 0.12 0.14 0.01 0.08 0.08 0.05 0.09 GI_34485729-S PRKY 0.02 0.21 0.14 0.07 0.03 0.05 0.09 0.09 GI_5031632-S FARP1 0.04 0.07 0.16 0.13 0.11 0.04 0.07 0.09 GI_33469973-A ATF4 0.02 0.10 0.20 0.04 0.04 0.03 0.20 0.09 GI_22095362-S C14orf135 0.02 0.21 0.23 0.00 0.03 0.03 0.09 0.09 GI_21361081-S CRLF1 0.10 0.15 0.09 0.09 0.04 0.12 0.03 0.09 GI_4557354-I BCL2 0.01 0.20 0.10 0.08 0.05 0.09 0.08 0.09 GI_45439305-S DARS 0.01 0.06 0.10 0.08 0.17 0.11 0.08 0.09 GI_31982935-S SGPL1 0.00 0.16 0.11 0.08 0.04 0.12 0.09 0.09 GI_8923891-S PXMP2 0.14 0.09 0.07 0.03 0.00 0.15 0.12 0.09 GI_7661645-S DKFZP566E144 0.08 0.13 0.13 0.02 0.10 0.03 0.12 0.09 GI_4503182-S CYB5 0.04 0.06 0.08 0.09 0.01 0.06 0.25 0.09 GI_15431291-S RPL12 0.01 0.09 0.08 0.04 0.01 0.13 0.24 0.09 GI_40255312-S P38IP 0.00 0.13 0.13 0.09 0.03 0.07 0.14 0.09 GI_31542585-S EIF4EBP2 0.04 0.11 0.06 0.04 0.12 0.16 0.05 0.08 GI_31343475-S GNA13 0.00 0.12 0.14 0.07 0.04 0.10 0.10 0.08 GI_24475891-S CSPG6 0.01 0.08 0.17 0.04 0.01 0.05 0.24 0.08 GI_42716292-S EMP2 0.02 0.16 0.05 0.12 0.11 0.02 0.11 0.08 GI_31543652-S SRP14 0.01 0.17 0.08 0.04 0.04 0.11 0.13 0.08 GI_25453469-S EEF1A1 0.04 0.08 0.08 0.07 0.02 0.09 0.19 0.08 GI_27436919-I SPAG9 0.03 0.11 0.13 0.04 0.07 0.06 0.14 0.08 GI_21359839-S SNRPG 0.01 0.06 0.10 0.08 0.01 0.12 0.19 0.08 GI_15451787-S PDGFRA 0.00 0.11 0.06 0.13 0.02 0.16 0.10 0.08 GI_19923602-S CYBRD1 0.01 0.17 0.13 0.03 0.07 0.10 0.05 0.08 GI_19743895-A TADA3L 0.10 0.04 0.11 0.06 0.01 0.12 0.12 0.08 GI_5730084-S TCTEL1 0.04 0.10 0.07 0.13 0.04 0.11 0.08 0.08 GI_15193293-S PGR1 0.13 0.00 0.12 0.07 0.01 0.02 0.22 0.08 GI_21704284-S JAM2 0.07 0.09 0.10 0.07 0.10 0.03 0.09 0.08 GI_4502370-S BCAR3 0.06 0.15 0.19 0.00 0.05 0.09 0.01 0.08 GI_37541013-S LOC374395 0.01 0.00 0.08 0.08 0.08 0.10 0.20 0.08 GI_34304355-A SCAPIN1 0.02 0.13 0.10 0.13 0.07 0.04 0.07 0.08 GI_21735593-I PDCD2 0.12 0.06 0.12 0.03 0.01 0.14 0.07 0.08 GI_23510344-I FYN 0.04 0.11 0.05 0.13 0.05 0.12 0.04 0.08 GI_7661537-S BRI3 0.12 0.14 0.07 0.06 0.05 0.03 0.08 0.08 GI_6806894-S PKP4 0.02 0.08 0.15 0.08 0.07 0.01 0.13 0.08 GI_41872597-S CPNE3 0.00 0.19 0.17 0.08 0.00 0.10 0.01 0.08 GI_41350211-S BRD7 0.00 0.07 0.11 0.05 0.00 0.08 0.22 0.08 GI_37542859-S DKFZp313M0720 0.06 0.13 0.07 0.08 0.03 0.08 0.10 0.08 GI_34304116-S UBC 0.05 0.03 0.07 0.06 0.03 0.05 0.23 0.08 GI_5174588-S MTF1 0.08 0.02 0.06 0.14 0.10 0.05 0.08 0.08 GI_21361584-S KIAA0992 0.01 0.17 0.08 0.09 0.04 0.12 0.01 0.08 GI_4503532-S EIF4B 0.03 0.09 0.10 0.11 0.02 0.01 0.17 0.08 GI_19263339-S GPT2 0.04 0.13 0.04 0.08 0.07 0.14 0.01 0.07 GI_40255140-S ChGn 0.20 0.10 0.06 0.01 0.03 0.10 0.02 0.07 GI_41406065-A H2AV 0.01 0.03 0.10 0.21 0.03 0.09 0.04 0.07 GI_30795205-S PPP2R5A 0.05 0.07 0.07 0.08 0.08 0.08 0.08 0.07 GI_31542744-S FLJ23091 0.06 0.14 0.04 0.09 0.02 0.12 0.03 0.07 GI_22538424-S ATPAF2 0.09 0.08 0.08 0.12 0.03 0.08 0.02 0.07 GI_24308042-S KIAA0828 0.04 0.08 0.09 0.07 0.08 0.11 0.02 0.07 GI_28316809-S MGC31967 0.02 0.07 0.08 0.06 0.06 0.02 0.19 0.07 GI_16905527-A DAP3 0.03 0.10 0.07 0.09 0.01 0.04 0.17 0.07 GI_22035601-A MAP4K4 0.01 0.07 0.06 0.14 0.08 0.02 0.12 0.07 GI_24497491-S SLC22A5 0.02 0.09 0.12 0.11 0.09 0.04 0.03 0.07 GI_21536323-A HNRPUL1 0.02 0.04 0.03 0.05 0.14 0.09 0.13 0.07 GI_40254815-S HSPCA 0.04 0.09 0.14 0.01 0.00 0.04 0.18 0.07 GI_32481212-S MK-STYX 0.05 0.03 0.15 0.02 0.05 0.05 0.16 0.07 GI_17402905-S RPL22 0.04 0.03 0.05 0.02 0.05 0.13 0.17 0.07 GI_28466988-S ATP10D 0.08 0.15 0.10 0.05 0.05 0.07 0.00 0.07 GI_10346134-S MAPRE2 0.03 0.04 0.13 0.04 0.06 0.03 0.16 0.07 GI_33946332-I ZC3HAV1 0.05 0.19 0.02 0.03 0.07 0.10 0.04 0.07 GI_41393560-S LAP3 0.03 0.07 0.08 0.11 0.03 0.05 0.11 0.07 GI_10863994-S ZNF410 0.03 0.11 0.10 0.07 0.07 0.05 0.08 0.07 GI_6715608-S MAPK4 0.07 0.12 0.07 0.08 0.03 0.06 0.05 0.07 GI_4505336-S NUBP1 0.06 0.09 0.04 0.06 0.10 0.05 0.08 0.07 GI_40353732-S NPM1 0.01 0.05 0.13 0.01 0.05 0.03 0.19 0.07 GI_5579480-S ARHN 0.06 0.05 0.09 0.01 0.05 0.00 0.21 0.07 GI_4503606-S ETFA 0.04 0.06 0.09 0.06 0.01 0.15 0.05 0.07 GI_34147357-S MGC2747 0.07 0.03 0.01 0.04 0.11 0.11 0.11 0.07 GI_4503174-S CXCR4 0.05 0.06 0.12 0.08 0.08 0.02 0.06 0.07 GI_37552472-S LOC286088 0.02 0.04 0.10 0.06 0.03 0.00 0.22 0.07 GI_24432092-S PHF13 0.01 0.08 0.12 0.02 0.07 0.09 0.08 0.07 GI_38683837-S CD47 0.01 0.06 0.12 0.11 0.04 0.02 0.11 0.07 GI_31543149-S MGC11308 0.01 0.05 0.10 0.13 0.06 0.02 0.09 0.07 GI_4501882-S ACTA2 0.02 0.05 0.01 0.11 0.16 0.06 0.05 0.07 GI_4557394-S CA2 0.04 0.10 0.10 0.01 0.03 0.04 0.14 0.07 GI_28872718-S BTG2 0.04 0.08 0.01 0.12 0.22 0.00 0.00 0.07 GI_45359860-S POLR2L 0.05 0.03 0.03 0.09 0.00 0.14 0.12 0.07 GI_17864091-S DNAH7 0.06 0.08 0.11 0.07 0.05 0.08 0.01 0.07 GI_20986484-S YAP1 0.01 0.15 0.06 0.03 0.03 0.14 0.04 0.07 GI_34147334-S FLJ20811 0.06 0.00 0.06 0.02 0.05 0.07 0.19 0.07 GI_22538460-S NCOR1 0.05 0.09 0.08 0.00 0.01 0.01 0.22 0.07 GI_15451934-I CDC14B 0.02 0.13 0.07 0.06 0.07 0.04 0.06 0.07 GI_4557378-S SERPING1 0.07 0.07 0.00 0.08 0.11 0.06 0.07 0.06 GI_29789284-S COMMD7 0.07 0.00 0.11 0.10 0.12 0.04 0.01 0.06 GI_4506342-S PXMP3 0.02 0.14 0.10 0.03 0.01 0.11 0.03 0.06 GI_24308106-S DKFZp566C0424 0.00 0.12 0.03 0.07 0.11 0.05 0.07 0.06 GI_34328906-I DNAJB6 0.04 0.07 0.10 0.07 0.06 0.03 0.08 0.06 GI_38373686-S AP1G1 0.05 0.02 0.12 0.06 0.08 0.07 0.05 0.06 GI_13654234-A RGS20 0.06 0.11 0.05 0.05 0.02 0.14 0.02 0.06 GI_4502100-S ANXA1 0.03 0.07 0.02 0.08 0.08 0.01 0.14 0.06 GI_34222274-S SSA2 0.05 0.11 0.08 0.06 0.11 0.01 0.01 0.06 GI_15011920-S NOLA3 0.12 0.02 0.06 0.05 0.04 0.06 0.08 0.06 GI_23510452-S COTL1 0.03 0.06 0.02 0.01 0.10 0.10 0.10 0.06 GI_23111020-A RGN 0.01 0.10 0.08 0.01 0.01 0.17 0.05 0.06 GI_32483398-S PAK2 0.07 0.04 0.13 0.12 0.01 0.03 0.04 0.06 GI_10835186-S SOD2 0.06 0.02 0.03 0.16 0.05 0.06 0.05 0.06 GI_23346408-S C20orf111 0.06 0.02 0.07 0.06 0.03 0.06 0.12 0.06 GI_5032214-S UK114 0.01 0.09 0.10 0.04 0.01 0.15 0.03 0.06 GI_39725690-S SPUF 0.02 0.13 0.05 0.05 0.05 0.02 0.11 0.06 GI_40255040-S TMP21 0.01 0.04 0.09 0.02 0.01 0.07 0.18 0.06 GI_39725692-S FLJ10420 0.01 0.07 0.03 0.05 0.07 0.08 0.12 0.06 GI_31542848-S GMPR 0.11 0.05 0.04 0.08 0.08 0.01 0.05 0.06 GI_24475893-S GNB2L1 0.02 0.03 0.02 0.05 0.02 0.08 0.19 0.06 GI_31581523-S COBL 0.02 0.15 0.11 0.06 0.02 0.03 0.03 0.06 GI_18375506-S APEX2 0.01 0.11 0.06 0.01 0.10 0.11 0.02 0.06 GI_34147490-S APG3 0.05 0.01 0.11 0.00 0.02 0.04 0.18 0.06 GI_39930611-I KLHL5 0.01 0.10 0.07 0.08 0.10 0.05 0.01 0.06 GI_42658769-S LOC401457 0.09 0.08 0.03 0.03 0.05 0.00 0.13 0.06 GI_40254977-S FIP1L1 0.01 0.06 0.05 0.06 0.10 0.03 0.10 0.06 GI_23510357-A RIOK1 0.04 0.06 0.14 0.05 0.02 0.02 0.07 0.06 GI_33946290-S FLJ12443 0.00 0.03 0.03 0.19 0.03 0.09 0.03 0.06 GI_21361092-S TPST1 0.15 0.05 0.00 0.02 0.01 0.08 0.10 0.06 GI_40254896-S DKFZp434K1210 0.05 0.10 0.02 0.08 0.05 0.05 0.05 0.06 GI_8922853-S FLJ11078 0.07 0.10 0.04 0.06 0.04 0.07 0.01 0.06 GI_8051633-S RARRES3 0.05 0.03 0.02 0.12 0.03 0.08 0.08 0.06 GI_31543826-I TSC22 0.03 0.09 0.12 0.01 0.03 0.00 0.12 0.06 GI_37694061-I AQP1 0.04 0.06 0.02 0.11 0.03 0.08 0.06 0.06 GI_19747274-A PHF10 0.02 0.16 0.03 0.07 0.08 0.02 0.01 0.06 GI_4557342-S ALDH7A1 0.02 0.06 0.04 0.09 0.01 0.15 0.02 0.06 GI_14043023-S BAG3 0.01 0.13 0.01 0.07 0.14 0.02 0.02 0.06 GI_18105013-A 3-Apr 0.00 0.04 0.02 0.03 0.07 0.10 0.13 0.06 GI_21618360-S FXYD5 0.04 0.06 0.02 0.08 0.09 0.02 0.09 0.06 GI_4503100-S CSRP2 0.03 0.09 0.06 0.05 0.07 0.02 0.08 0.06 GI_23308566-S ASRGL1 0.05 0.10 0.01 0.11 0.02 0.08 0.02 0.06 GI_42658538-S DKFZP434A0225 0.03 0.03 0.06 0.13 0.09 0.02 0.03 0.06 GI_40255086-S LOC118491 0.04 0.05 0.10 0.02 0.07 0.06 0.06 0.06 GI_7661691-S DKFZP586N0721 0.09 0.13 0.00 0.07 0.02 0.01 0.05 0.06 GI_4757755-S ANXA2 0.01 0.07 0.04 0.08 0.10 0.01 0.06 0.05 GI_14249551-S DIRC2 0.06 0.09 0.05 0.05 0.08 0.02 0.04 0.05 GI_20070179-S EIF4EBP1 0.02 0.07 0.05 0.14 0.04 0.01 0.05 0.05 GI_4503680-S FCGBP 0.01 0.06 0.05 0.03 0.08 0.03 0.12 0.05 GI_41188450-S LOC388727 0.01 0.04 0.01 0.15 0.08 0.08 0.01 0.05 GI_42717993-A DTNA 0.04 0.08 0.06 0.06 0.02 0.05 0.08 0.05 GI_4505488-S ODC1 0.10 0.10 0.03 0.00 0.04 0.05 0.05 0.05 GI_19923436-S AK3L1 0.04 0.06 0.01 0.02 0.10 0.13 0.00 0.05 GI_31341159-S MGC21416 0.15 0.02 0.06 0.00 0.11 0.01 0.00 0.05 GI_8922937-S FLJ11200 0.00 0.07 0.10 0.05 0.05 0.06 0.03 0.05 GI_34222296-S FCGRT 0.01 0.08 0.02 0.07 0.07 0.09 0.02 0.05 GI_32307151-S OXTR 0.11 0.04 0.02 0.04 0.03 0.04 0.07 0.05 GI_34222132-S TXNDC 0.04 0.07 0.06 0.04 0.04 0.09 0.02 0.05 GI_22035637-A MGST1 0.01 0.09 0.01 0.06 0.02 0.13 0.03 0.05 GI_34147669-S SLC39A1 0.02 0.04 0.04 0.04 0.13 0.02 0.04 0.05 GI_21071079-S FBXL7 0.00 0.09 0.03 0.06 0.06 0.08 0.01 0.05 GI_31543361-S ORF1-FL49 0.05 0.08 0.02 0.07 0.01 0.10 0.00 0.05 GI_42716312-S ANG 0.01 0.02 0.05 0.06 0.08 0.09 0.02 0.05 GI_6006016-S LGALS3BP 0.01 0.05 0.03 0.10 0.06 0.02 0.06 0.05 GI_40549455-S NHS 0.01 0.13 0.05 0.03 0.04 0.04 0.03 0.05 GI_7019466-S CNOT4 0.02 0.12 0.09 0.05 0.00 0.03 0.02 0.05 GI_24797094-A PYCR1 0.01 0.13 0.06 0.07 0.03 0.01 0.02 0.05 GI_7657057-S EIF2B2 0.02 0.06 0.05 0.02 0.03 0.10 0.05 0.05 GI_20127485-S M6PRBP1 0.00 0.01 0.00 0.10 0.11 0.04 0.06 0.05 GI_4755145-S AEBP1 0.01 0.02 0.02 0.12 0.08 0.01 0.05 0.04 GI_33943097-S RAB5B 0.07 0.00 0.06 0.07 0.09 0.01 0.00 0.04 GI_16445423-S WDR12 0.04 0.08 0.04 0.02 0.02 0.02 0.08 0.04 GI_42476129-S RAP80 0.04 0.03 0.07 0.07 0.06 0.03 0.01 0.04 GI_34147349-S MGC2601 0.03 0.03 0.03 0.03 0.08 0.08 0.02 0.04 GI_29789057-S KIBRA 0.04 0.06 0.01 0.00 0.04 0.11 0.03 0.04 GI_14670391-A BAZ1B 0.04 0.00 0.07 0.00 0.12 0.01 0.04 0.04 GI_4557800-S NP 0.04 0.01 0.02 0.10 0.02 0.06 0.03 0.04 GI_34147469-S MGC15396 0.02 0.08 0.03 0.03 0.07 0.02 0.05 0.04 GI_13129101-S MGC955 0.04 0.00 0.02 0.10 0.02 0.06 0.03 0.04 GI_39753966-S CSPG5 0.00 0.02 0.04 0.03 0.00 0.08 0.11 0.04 GI_33636718-S TIMM44 0.04 0.03 0.05 0.02 0.07 0.04 0.02 0.04 GI_40354215-S SIX5 0.00 0.10 0.02 0.07 0.04 0.05 0.00 0.04 GI_18702322-S DNCL2B 0.02 0.10 0.06 0.02 0.04 0.02 0.01 0.04 GI_20127480-S EPM2A 0.08 0.02 0.00 0.04 0.06 0.05 0.02 0.04 GI_18104963-A CAPS 0.00 0.05 0.01 0.05 0.10 0.03 0.02 0.04 GI_4507432-S TEGT 0.04 0.04 0.01 0.04 0.08 0.06 0.00 0.04 GI_24586683-A DMN 0.02 0.07 0.06 0.00 0.07 0.02 0.02 0.04 GI_20149303-S KIAA1160 0.09 0.03 0.03 0.03 0.00 0.02 0.05 0.04 GI_4557586-S FAH 0.06 0.01 0.03 0.05 0.04 0.07 0.01 0.04 GI_11496992-S ADPRTL3 0.07 0.06 0.03 0.04 0.04 0.01 0.00 0.04 GI_37550187-S LOC375468 0.01 0.12 0.01 0.02 0.00 0.01 0.07 0.04 GI_34222312-S GPSM2 0.01 0.09 0.02 0.04 0.01 0.01 0.05 0.03 GI_34147344-S KCTD14 0.04 0.08 0.08 0.03 0.00 0.00 0.01 0.03 GI_7706271-S CGI-30 0.07 0.06 0.02 0.00 0.01 0.07 0.01 0.03 GI_34147623-S SORD 0.02 0.04 0.09 0.02 0.02 0.04 0.01 0.03 GI_40255159-S MGC20446 0.04 0.06 0.06 0.01 0.00 0.04 0.01 0.03 GI_19311011-S BRIX 0.00 0.04 0.04 0.01 0.01 0.02 0.10 0.03 GI_24797087-A PEX10 0.01 0.02 0.00 0.00 0.02 0.12 0.05 0.03 GI_20336263-A GGA2 0.06 0.03 0.01 0.02 0.02 0.06 0.02 0.03 GI_21361710-S HCNGP 0.02 0.00 0.02 0.00 0.02 0.04 0.03 0.02 GI_31543911-S USP20 −0.02 −0.11 −0.16 −0.12 −0.14 −0.04 −0.16 −0.11 GI_5902039-S RABL2B −0.01 −0.04 −0.05 −0.25 −0.15 −0.06 −0.14 −0.10 GI_32454736-A TRIM3 −0.12 −0.02 −0.13 −0.12 −0.13 −0.02 −0.14 −0.10 GI_18598508-S CDR2 −0.02 −0.07 −0.09 −0.18 −0.14 −0.10 −0.08 −0.10 GI_7549818-A RABL2A −0.04 −0.02 −0.05 −0.23 −0.16 −0.05 −0.13 −0.10 GI_14149994-S DKFZp434N035 −0.02 −0.13 −0.18 −0.07 −0.11 −0.06 −0.11 −0.10 GI_37577147-A NCKIPSD −0.07 −0.06 −0.11 −0.11 −0.11 −0.04 −0.17 −0.10 GI_34147578-S PISD −0.06 −0.02 −0.14 −0.14 −0.13 −0.08 −0.09 −0.10 GI_4505328-S NAPA −0.09 −0.07 −0.21 −0.04 −0.03 −0.10 −0.11 −0.09 GI_13162281-S STS −0.02 −0.15 −0.10 −0.13 −0.04 −0.09 −0.11 −0.09 GI_21450766-S C6orf136 −0.05 −0.12 −0.10 −0.10 −0.10 −0.07 −0.09 −0.09 GI_11641403-S CKMT1 −0.05 −0.17 −0.08 −0.08 −0.10 −0.07 −0.06 −0.09 GI_33636749-S E2-230K 0.00 −0.12 −0.12 −0.08 0.00 −0.16 −0.11 −0.09 GI_37577121-I UBE2J1 −0.07 −0.07 −0.11 −0.11 −0.07 −0.01 −0.17 −0.09 GI_31377702-S TTC13 −0.06 −0.13 −0.05 −0.09 −0.12 −0.06 −0.08 −0.09 GI_32528302-S INSM2 −0.05 −0.07 −0.18 −0.17 −0.02 −0.02 −0.09 −0.08 GI_14149741-S KIAA1536 −0.08 −0.11 −0.10 −0.06 −0.09 −0.04 −0.10 −0.08 GI_34222158-S FLJ10925 −0.07 −0.14 −0.09 −0.15 −0.03 0.00 −0.09 −0.08 GI_8923764-S CACNA2D3 −0.01 −0.17 −0.06 −0.04 −0.05 −0.08 −0.15 −0.08 GI_28372510-S ZDHHC22 −0.02 −0.14 −0.12 −0.14 −0.11 −0.03 0.00 −0.08 GI_21735551-S MAP3K12 −0.08 −0.12 −0.12 −0.02 −0.03 −0.01 −0.16 −0.08 GI_21361926-S C6orf31 −0.06 −0.03 −0.11 −0.05 −0.14 −0.05 −0.11 −0.08 GI_22095352-S BCAS3 −0.14 −0.11 −0.17 0.00 −0.01 0.00 −0.10 −0.08 GI_24430154-A PSMC4 −0.06 −0.10 −0.14 −0.08 −0.10 0.00 −0.06 −0.08 GI_24307956-S PIB5PA −0.04 −0.03 −0.09 −0.14 −0.04 −0.05 −0.14 −0.08 GI_29540546-A TRO −0.10 −0.08 −0.03 −0.10 −0.04 −0.05 −0.13 −0.08 GI_32454751-S ORC2L −0.07 −0.11 −0.06 −0.12 −0.04 −0.05 −0.08 −0.08 GI_37588868-S RNF123 −0.06 −0.01 −0.15 −0.06 −0.03 −0.09 −0.13 −0.08 GI_22538494-S WBSCR17 −0.07 −0.09 −0.12 −0.01 −0.11 −0.06 −0.07 −0.08 GI_14149656-S KIAA1049 −0.01 −0.15 −0.13 −0.06 −0.05 −0.06 −0.06 −0.07 GI_23618866-S SFXN1 −0.10 −0.01 −0.04 −0.08 −0.07 −0.01 −0.22 −0.07 GI_31342415-S LOC90529 −0.01 −0.06 −0.08 −0.09 −0.09 −0.08 −0.12 −0.07 GI_17017983-S CDK9 −0.10 −0.10 −0.05 −0.05 −0.04 −0.03 −0.15 −0.07 GI_21536352-S ACTL6 −0.01 −0.12 −0.13 −0.08 −0.06 −0.01 −0.09 −0.07 GI_38158008-A CIDEA −0.01 −0.17 −0.02 −0.05 −0.10 −0.04 −0.13 −0.07 GI_20336241-S PCSK1 −0.04 −0.09 −0.09 −0.12 −0.07 −0.07 −0.03 −0.07 GI_19557635-A PPIL3 −0.02 −0.01 −0.11 −0.12 −0.01 −0.11 −0.13 −0.07 GI_13376446-S C20orf98 −0.02 −0.07 −0.17 −0.02 −0.08 0.00 −0.14 −0.07 GI_14042940-S eIF2A −0.01 −0.14 −0.04 −0.14 −0.01 −0.08 −0.09 −0.07 GI_27436965-A KCNAB1 −0.10 −0.05 −0.09 −0.07 −0.09 −0.03 −0.08 −0.07 GI_4757805-S C16orf7 −0.06 −0.10 −0.15 −0.05 −0.02 −0.02 −0.10 −0.07 GI_7662423-S KIAA0972 −0.08 −0.08 −0.02 −0.18 −0.02 −0.07 −0.06 −0.07 GI_34303930-S MGC20262 −0.10 −0.01 −0.11 −0.09 −0.01 −0.02 −0.16 −0.07 GI_4758897-A PEX16 −0.07 −0.11 −0.20 −0.04 −0.02 0.00 −0.06 −0.07 GI_19923722-S RPS6KC1 −0.01 −0.09 −0.10 −0.09 −0.07 −0.05 −0.10 −0.07 GI_40255224-S FLJ12892 −0.05 −0.01 −0.02 −0.15 −0.10 −0.04 −0.10 −0.07 GI_34222360-S ATP1A1 −0.02 −0.11 −0.11 −0.02 −0.07 −0.09 −0.05 −0.07 GI_5454157-S VARS2 −0.04 −0.14 −0.14 −0.04 −0.02 −0.09 −0.02 −0.07 GI_31657108-S ZNF282 −0.04 −0.13 −0.04 −0.03 −0.02 −0.18 −0.04 −0.07 GI_16933538-A GLMN −0.03 −0.07 −0.02 −0.10 −0.11 −0.01 −0.15 −0.07 GI_41327772-S DDX46 −0.13 −0.07 −0.09 −0.05 −0.05 −0.03 −0.06 −0.07 GI_24431993-S MGC3234 −0.06 −0.07 −0.06 −0.07 −0.03 −0.05 −0.13 −0.07 GI_19718776-S FEN1 −0.01 −0.12 −0.07 −0.07 −0.08 −0.07 −0.05 −0.07 GI_38026914-A ARHGEF11 0.00 −0.04 −0.10 −0.05 −0.08 −0.15 −0.04 −0.07 GI_38570137-S MGC15677 −0.11 −0.09 −0.11 −0.04 0.00 −0.06 −0.06 −0.07 GI_34222197-S C10orf22 −0.07 −0.03 −0.01 −0.07 −0.07 −0.09 −0.12 −0.07 GI_12232402-S FLJ13868 −0.07 −0.02 −0.05 −0.09 −0.10 −0.01 −0.12 −0.07 GI_13899304-S CD99L2 −0.06 −0.05 −0.13 −0.01 −0.05 −0.07 −0.09 −0.07 GI_31543933-S VMP −0.05 −0.07 −0.12 −0.06 −0.06 −0.03 −0.08 −0.07 GI_13376430-S FLJ13397 −0.02 −0.04 −0.04 −0.09 −0.10 −0.03 −0.13 −0.07 GI_7669496-S JWA −0.04 −0.04 −0.07 −0.04 −0.11 −0.02 −0.14 −0.07 GI_34147471-S MGC20781 −0.01 −0.10 −0.11 −0.12 −0.05 −0.02 −0.06 −0.06 GI_41146823-S LOC389197 −0.10 −0.06 −0.07 −0.06 −0.08 −0.02 −0.06 −0.06 GI_42734386-S LOC199692 −0.03 0.00 −0.04 −0.04 −0.12 −0.03 −0.20 −0.06 GI_21362099-S ELOVL4 −0.03 −0.10 −0.01 −0.08 −0.06 −0.05 −0.13 −0.06 GI_18105052-S RAE1 −0.05 −0.08 −0.07 −0.02 −0.08 −0.11 −0.03 −0.06 GI_34222118-S SYT4 −0.10 −0.06 0.00 −0.05 −0.12 −0.04 −0.08 −0.06 GI_34147419-S ACBD6 0.00 −0.18 −0.08 −0.06 −0.06 −0.02 −0.05 −0.06 GI_19923214-S MEF2C −0.02 −0.06 −0.07 −0.07 −0.08 −0.02 −0.11 −0.06 GI_4758181-S DNM1 −0.08 −0.09 −0.12 −0.01 −0.05 0.00 −0.08 −0.06 GI_22748930-S FBXL14 −0.05 −0.02 −0.03 −0.07 −0.05 −0.02 −0.20 −0.06 GI_7706644-S PME-1 −0.04 −0.12 −0.08 −0.02 −0.08 −0.06 −0.03 −0.06 GI_6466453-S SNCB −0.10 −0.09 −0.07 −0.01 −0.11 −0.05 −0.02 −0.06 GI_21361484-S DKFZP434P1750 −0.06 −0.13 −0.12 −0.02 −0.05 −0.04 −0.03 −0.06 GI_7657503-S RBM9 −0.02 −0.11 −0.03 −0.07 −0.04 −0.07 −0.09 −0.06 GI_19718745-A OSBPL1A −0.03 −0.04 −0.10 −0.07 −0.04 −0.08 −0.07 −0.06 GI_18373307-S RAB40C 0.00 −0.06 −0.11 −0.04 −0.02 −0.10 −0.10 −0.06 GI_33598918-A SCAMP1 −0.04 −0.02 −0.06 −0.06 −0.02 −0.08 −0.14 −0.06 GI_21361102-S SLC25A12 0.00 −0.09 −0.02 −0.14 −0.07 −0.03 −0.07 −0.06 GI_8922070-S LOC55565 −0.04 −0.06 −0.10 0.00 −0.05 0.00 −0.16 −0.06 GI_4507212-S SRP19 −0.05 −0.06 −0.05 −0.08 −0.03 −0.01 −0.14 −0.06 GI_40068504-S BSCL2 −0.01 −0.12 −0.08 −0.01 −0.08 −0.04 −0.08 −0.06 GI_13027629-S DGCR14 −0.09 −0.11 −0.14 −0.04 0.00 −0.01 −0.01 −0.06 GI_9257239-A SDFR1 −0.05 −0.03 −0.02 −0.03 −0.13 −0.02 −0.14 −0.06 GI_25121973-S LOC151835 −0.06 0.00 −0.04 −0.17 −0.03 −0.08 −0.02 −0.06 GI_5453861-S PDE4A −0.03 −0.09 −0.09 −0.12 −0.03 −0.02 −0.02 −0.06 GI_34222152-S VSNL1 −0.14 −0.03 −0.02 −0.02 −0.09 −0.02 −0.08 −0.06 GI_32490571-S EPB41L3 −0.06 −0.10 −0.03 −0.04 −0.03 −0.12 −0.03 −0.06 GI_13325063-S CELSR2 −0.02 −0.04 −0.06 −0.11 −0.06 −0.09 −0.02 −0.06 GI_27764866-S SYP −0.01 −0.11 −0.08 −0.04 −0.11 −0.03 −0.01 −0.06 GI_37622344-A ZNF42 −0.06 −0.05 −0.05 −0.05 0.00 −0.05 −0.12 −0.06 GI_10092616-S PCBP3 −0.02 −0.06 −0.06 −0.06 −0.10 −0.01 −0.06 −0.06 GI_31343339-S FLJ33996 0.00 −0.12 −0.03 −0.07 −0.05 −0.08 −0.04 −0.06 GI_13375816-S NEIL1 −0.04 −0.01 −0.08 −0.16 −0.01 −0.01 −0.08 −0.05 GI_18550284-S KIAA1912 −0.03 −0.01 −0.03 −0.05 −0.11 −0.03 −0.13 −0.05 GI_4507104-S SNAPC3 −0.01 −0.04 −0.05 −0.11 −0.09 −0.05 −0.02 −0.05 GI_20127649-S KIAA0157 −0.04 −0.02 0.00 −0.06 −0.07 −0.06 −0.12 −0.05 GI_35493938-S ProSAPiP1 −0.01 −0.05 −0.09 −0.03 −0.01 −0.08 −0.12 −0.05 GI_33667083-S DNAJC9 −0.01 −0.02 −0.04 −0.10 −0.12 −0.05 −0.05 −0.05 GI_37555997-S LOC375663 −0.09 −0.01 −0.07 −0.08 −0.03 0.00 −0.09 −0.05 GI_22748944-S MGC26690 −0.04 −0.07 −0.08 −0.04 0.00 −0.01 −0.13 −0.05 GI_39725643-A MR-1 −0.01 −0.11 −0.07 −0.06 −0.03 −0.07 −0.03 −0.05 GI_42734431-S NLK −0.04 −0.03 −0.05 −0.08 −0.03 −0.09 −0.04 −0.05 GI_4502286-S ATP2B1 −0.09 −0.02 −0.08 −0.05 −0.01 −0.04 −0.07 −0.05 GI_33391149-S NPM2 −0.03 −0.06 −0.07 −0.09 −0.06 −0.01 −0.04 −0.05 GI_44955932-I UBQLN1 −0.06 0.00 0.00 −0.08 −0.06 −0.10 −0.07 −0.05 GI_24308367-S FLJ38944 −0.01 −0.09 −0.10 −0.05 −0.04 0.00 −0.07 −0.05 GI_37059735-S CWF19L1 −0.03 −0.07 −0.01 −0.08 0.00 −0.08 −0.08 −0.05 GI_24797146-S SEPHS2 −0.05 −0.07 −0.05 −0.04 −0.06 −0.04 −0.05 −0.05 GI_42476122-S RUSC1 −0.01 −0.04 −0.03 −0.04 −0.08 −0.04 −0.10 −0.05 GI_24307960-S KIAA0406 −0.01 −0.04 −0.05 −0.06 −0.02 −0.02 −0.14 −0.05 GI_40354211-S PIP3-E −0.06 −0.08 −0.01 −0.09 −0.03 −0.02 −0.05 −0.05 GI_34147620-A AMPD2 −0.03 −0.03 −0.09 −0.03 −0.06 −0.02 −0.08 −0.05 GI_21071040-S CNTNAP2 −0.02 −0.06 −0.04 −0.07 −0.05 −0.05 −0.04 −0.05 GI_27436982-S KCND2 −0.03 −0.04 −0.04 −0.04 −0.09 −0.02 −0.09 −0.05 GI_29029532-A SULT4A1 −0.01 −0.13 −0.03 −0.05 −0.06 −0.06 0.00 −0.05 GI_4507830-S ULK1 0.00 −0.03 −0.10 −0.04 −0.03 0.00 −0.13 −0.05 GI_32698821-S LOC90637 −0.10 −0.04 −0.02 −0.05 −0.04 −0.04 −0.05 −0.05 GI_11968046-S PAF53 −0.03 −0.05 −0.04 0.00 −0.08 −0.01 −0.12 −0.05 GI_45359844-S G3BP2 −0.04 −0.06 −0.03 −0.03 −0.12 −0.01 −0.04 −0.05 GI_34101281-S SCNN1D −0.01 −0.06 −0.03 −0.07 −0.03 −0.05 −0.06 −0.05 GI_34147584-S DMAP1 −0.03 −0.07 −0.06 −0.04 −0.01 −0.03 −0.08 −0.04 GI_37537698-S LOC147965 −0.03 −0.05 −0.07 −0.05 −0.01 −0.02 −0.06 −0.04 GI_24432025-S FLJ14360 0.00 −0.02 −0.10 −0.01 −0.01 −0.02 −0.14 −0.04 GI_8922197-S FLJ10038 0.00 −0.13 −0.03 −0.06 −0.01 −0.02 −0.05 −0.04 GI_10938009-A TSC2 −0.07 −0.05 −0.07 −0.08 −0.01 −0.01 0.00 −0.04 GI_24308110-S DKFZp564O1863 −0.05 −0.03 −0.04 −0.07 −0.07 −0.01 −0.02 −0.04 GI_4826693-S DGCR2 −0.04 −0.06 −0.10 0.00 −0.03 −0.02 −0.02 −0.04 GI_22035549-S APBA2 −0.05 −0.08 −0.01 −0.02 −0.09 −0.01 −0.01 −0.04 GI_19923443-S CGI-141 −0.03 0.00 −0.04 −0.09 −0.08 −0.02 −0.01 −0.04 GI_30795206-S PPP2R5B −0.05 −0.05 −0.08 −0.05 −0.04 0.00 0.00 −0.04 GI_22027545-S CACNG3 −0.05 −0.01 −0.03 −0.02 −0.07 −0.04 −0.04 −0.04 GI_23065565-S GPR24 −0.01 −0.03 −0.05 −0.04 −0.02 −0.09 −0.03 −0.04 GI_45439315-I PPIE −0.02 −0.09 −0.03 −0.01 −0.04 −0.05 −0.01 −0.04 GI_40255102-S ZNF488 −0.05 −0.09 −0.03 −0.01 0.00 −0.05 −0.01 −0.03 GI_4505460-S ENC1 −0.03 −0.04 0.00 −0.03 −0.07 −0.03 −0.04 −0.03 GI_30795230-S BASP1 −0.01 −0.02 −0.01 −0.02 −0.06 −0.05 −0.06 −0.03 GI_4758403-S FRG1 −0.03 −0.03 −0.02 −0.02 −0.04 −0.03 −0.03 −0.03 GI_45439343-I PPIL5 −0.02 −0.02 −0.04 −0.06 0.00 −0.02 −0.03 −0.03 GI_16445028-S IGSF8 −0.05 0.00 −0.07 −0.01 0.00 −0.02 −0.04 −0.03 GI_19913415-A AP2A1 0.00 −0.02 −0.03 −0.02 −0.02 −0.06 −0.04 −0.03 GI_29126235-S PGSF1 −0.02 −0.01 −0.03 −0.01 0.00 −0.05 −0.01 −0.02

This observation of an overlapping GPI for these 7 PD-associated loci was moreover confirmed in an additional independent dataset of cerebral frontal cortex autopsy brain tissue of 143 individuals (p=1.6 E-3 by resampling statistics; derived from GEO GSE15745).

Function annotation was performed on the gene expression changes that underlie the common GPIs among PD risk variants. Strikingly, among the annotated gene sets most significantly reduced in expression are “mitochondria” functions (FIGS. 8C-8D), consistent with the well-described association of defects in mitochondria with PD pathology (Zheng et al., 2010). Furthermore, the common overlapping transcriptomic signature of gene expression changes associated with these 7 PD risk variants revealed a pattern most similar to the transcriptome changes observed in the context of PD patient brain tissue (relative to unaffected brain tissue; FIG. 8C), rather than to other CNS disorders such as Alzheimer's disease or schizophrenia.

LRRK2 and PARK16 Variants Cooperatively Determine PD Risk

Among the 7 analyzed PD risk locus GPIs, those at the PARK16 and LRRK2 loci were found to be the most similar. Furthermore, variants at these two loci impacted the transcriptome in a non-additive manner, signifying a genetic interaction (as determined by analysis of carriers of both risk variants; FIG. 1D). It was investigated whether these loci similarly genetically interact in terms of their impact on PD risk: namely, whether harboring either a risk (or protective) allele at one of these loci would modify the association of the second locus with PD risk. In an initial study on an Ashkenazi Jewish (AJ) population, the effect of a risk-associated variant at the LRRK2 locus was in fact highly dependent on the presence of the risk variant at the PARK16 locus, and vice versa (FIG. 1E). Such ‘epistasis’ between the LRRK2 and PARK16 loci regarding PD risk was replicated by reanalysis of 3 other independent GWAS, strongly supporting a common mechanism of action (FIG. 1E). Although prior studies have not reported genetic interactions with the common sporadic PD risk-associated variants at the LRRK2 locus, a GWAS of patients who harbor rare familial LRRK2 mutations identified a broad 15 Mb region of Chromosome 1 as harboring a genetic modifier of age of PD onset (Latourelle et al., 2011). It is noted that this region encompassed the PARK16 locus. Meta-analysis in 4 independent sporadic PD GWAS datasets (as above) of the 74 identified SNP variants within this Chromosome 1 region for epistasis with the common LRRK2 SNP variant regarding PD risk identified the PARK16-associated variant as by far the most significantly interacting variant (combined p-value for epistasis: 4.6E-6; FIG. 1F, Table 3).

TABLE 3 LRRK2-PARK16 epistasis meta-analysis. NGRC NINDS SNP Chr1 pos. SNP Int. OR p SNP Int. OR p rs12063329 148269060 rs12063329 0.95 0.6179 rs12063329 0.82 0.4924 rs6684514 154522080 rs6984514 1.04 0.6500 rs6684514 0.97 0.8855 rs10908495 154530564 rs10908495 1.04 0.6220 rs10908495 0.96 0.8442 rs10908496 154530624 rs10908496 1.04 0.6428 rs10908496 0.96 0.8601 rs10908498 154544799 rs10908498 1.04 0.6140 rs10908498 0.96 0.8442 rs10908502 154566706 rs10908502 1.04 0.6528 rs10908502 0.96 0.8442 rs2789425 158216275 rs2789424 1.09 0.2855 rs2789425 0.96 0.8509 rs2737703 158322556 rs2737703 1.02 0.8052 rs2737703 1.69 0.0218 rs2369406 158331042 rs2369406 0.92 0.2705 rs2369406 0.72 0.1424 rs12057296 163609587 rs12057296 1.02 0.7676 rs12057296 1.37 0.1456 rs3767443 165787088 rs3767443 1.14 0.0841 rs3767443 0.84 0.4414 rs10489248 169912212 rs10489248 0.95 0.6108 rs10489248 1.22 0.5113 rs2014613 169914660 rs10489248 0.95 0.6108 rs2014613 1.19 0.5730 rs3753539 169982808 rs10753181 0.93 0.3902 rs3753539 1.10 0.7107 rs10912977 173565736 rs12565878 0.99 0.9015 rs10912977 0.76 0.2614 rs4652143 174250617 rs4652143 1.04 0.7926 rs4652143 1.17 0.7223 rs11587254 174420189 rs11579181 1.11 0.5384 rs4652143 1.17 0.7223 rs2294254 175445331 rs989536 0.99 0.9413 rs2294254 1.08 0.7621 rs946817 176264332 rs946817 1.06 0.4805 rs946817 1.19 0.4904 rs1281323 180368363 rs1281336 1.02 0.7906 rs1281323 0.91 0.6719 rs2254327 180490378 rs6662373 1.00 0.9916 rs2254327 1.57 0.0487 rs10911659 183171884 rs10911659 0.93 0.3234 rs10911659 0.91 0.6539 rs234092 183186797 rs234092 0.92 0.3495 rs234092 1.09 0.7840 rs234095 183197652 rs234098 0.95 0.5392 rs234095 1.03 0.9132 rs6684195 183293436 rs2378957 1.09 0.2687 rs6684195 1.01 0.9704 rs1200610 183418001 rs1200610 0.96 0.6659 rs1200610 1.14 0.6486 rs1208517 183539106 rs1208517 1.00 0.9810 rs1208517 1.06 0.8244 rs10489485 183605173 rs10489486 1.04 0.6859 rs10489486 0.88 0.6173 rs2186024 190696708 rs2186024 1.07 0.3788 rs1286024 0.81 0.3315 rs2494354 192721321 rs4427392 0.96 0.6875 rs7515494 1.25 0.5291 rs2494312 192763213 rs4427392 0.96 0.6875 rs2494312 1.21 0.5771 rs927724 194441413 rs927724 1.08 0.5087 rs2094026 2.04 0.0601 rs599779 197604771 rs576141 0.91 0.1984 rs599779 1.12 0.5986 rs1898240 197613703 rs1898240 0.93 0.4634 rs1898240 0.96 0.8838 rs487359 197648234 rs571754 0.91 0.2021 rs577752 0.95 0.8005 rs1890133 197721501 rs1890133 0.96 0.6363 rs1890133 1.42 0.1824 rs1400875 200088066 rs2820295 0.97 0.6978 rs1400875 1.26 0.3257 rs2820312 200135880 rs2820312 0.97 0.7299 rs2820312 1.23 0.3759 rs2050935 201598170 rs1977812 0.90 0.2352 rs2050935 0.78 0.3073 rs4950978 203291196 rs1470637 1.07 0.3564 rs4950978 0.97 0.8878 rs1470637 203299906 rs3851287 1.08 0.4049 rs1470637 1.13 0.5737 rs873114 203986155 rs873114 0.84 0.0117 rs873114 0.81 0.0099 rs2802221 205643068 rs2651361 1.18 0.0289 rs2802221 0.74 0.1727 rs643930 206224964 rs560311 0.97 0.6541 rs643930 0.93 0.7161 rs10729481 206755035 rs11119079 1.08 0.6451 rs10779481 1.15 0.8361 rs11119078 206758345 rs11119079 1.08 0.6451 rs11119078 1.23 0.7245 rs1933564 207098611 rs1933564 1.06 0.4926 rs1933564 1.21 0.3966 rs11119439 208447880 rs591594 0.97 0.7184 rs11119439 0.96 0.8673 rs590152 208460541 rs591594 0.97 0.7184 rs590152 0.96 0.8673 rs12124008 209710495 rs12124008 1.17 0.0333 rs12124008 0.79 0.2615 rs3104209 211172801 rs3124669 1.18 0.0225 rs3104209 1.04 0.8567 rs487208 213209901 rs1452632 0.90 0.1258 rs487208 0.76 0.1663 rs7548730 213911770 rs7548730 1.05 0.4447 rs7548730 1.09 0.7149 rs7547186 215003504 rs7547186 1.05 0.4928 rs7547186 1.02 0.9243 rs10495064 215896812 rs10495064 1.16 0.1339 rs10495064 1.63 0.1077 rs6673733 215982980 rs6673733 0.95 0.4659 rs6673733 0.75 0.1760 rs2377781 216350745 rs2377781 1.07 0.3383 rs2377781 1.25 0.3612 rs9441867 220044136 rs4433403 1.07 0.4005 rs9441867 0.74 0.2178 rs1341331 224702683 rs12118824 0.98 0.7591 rs1341331 1.17 0.4648 rs750426 224715286 rs4653740 1.04 0.6729 rs250426 0.78 0.3220 rs898833 224737622 rs898833 1.03 0.7660 rs898833 0.81 0.4035 rs11122571 228888383 rs11122571 0.92 0.2962 rs11122571 1.24 0.3892 rs7540252 228949072 rs7540252 1.04 0.6123 rs7540252 0.76 0.2078 rs1316408 228952336 rs1316408 1.01 0.8658 rs1316408 1.20 0.4449 rs7542797 229620410 rs7542797 1.19 0.0763 rs7542797 0.93 0.7858 rs487770 232843716 rs621901 0.86 0.0944 rs487770 0.95 0.8613 rs12746334 233775161 rs12746334 1.27 0.0768 rs12746334 1.20 0.6337 rs12135445 237809880 rs12135445 0.99 0.9037 rs12135445 0.89 0.6888 rs879081 238706181 rs879081 1.05 0.5440 rs879081 1.35 0.2019 rs9287247 238713161 rs9287247 0.97 0.6916 rs9287247 0.74 0.1831 rs2066380 238968131 rs2066380 0.86 0.0854 rs2066380 0.82 0.3853 rs9661248 243001296 rs9661248 0.98 0.8497 rs9661248 1.33 0.3027 rs4654274 244735167 rs4654274 1.02 0.8553 rs4654274 0.98 0.9330 AJ MAYO Combined SNP Int. OR p SNP Int. OR p p rs12063329 0.94 0.8662 rs2039800 1.02 0.9480 0.5194 rs6684514 0.67 0.1444 rs11264467 0.86 0.5694 0.3901 rs10908495 0.67 0.1444 rs11264467 0.86 0.5694 0.3865 rs10908496 0.67 0.1444 rs11264467 0.86 0.5694 0.3840 rs10908498 0.67 0.1444 rs11264467 0.86 0.5694 0.3896 rs10908502 0.67 0.1444 rs11264467 0.86 0.5694 0.3748 rs2789425 1.10 0.7236 rs2789425 0.78 0.4531 0.8090 rs2737703 1.09 0.7647 rs7512587 0.83 0.5479 0.2630 rs2369406 1.27 0.3857 rs2369406 1.09 0.7563 0.4867 rs12057296 1.00 0.9895 rs6675585 0.75 0.3243 0.7069 rs3767443 1.05 0.8429 rs1229430 1.09 0.7504 0.4612 rs10489248 0.92 0.7340 rs10913508 1.67 0.0889 0.4505 rs2014613 0.91 0.7030 rs10913508 1.67 0.0889 0.4919 rs3753539 0.89 0.6261 rs11808099 1.27 0.5313 0.8613 rs10912977 0.75 0.3376 rs6656777 0.71 0.3228 0.1102 rs4652143 1.03 0.9589 rs2502841 0.81 0.6137 0.9342 rs11587254 1.13 0.8516 rs17351808 1.13 0.7798 0.4763 rs2294254 0.91 0.7554 rs12758344 1.28 0.4058 0.7081 rs946817 0.96 0.9041 rs7519563 0.96 0.8925 0.5688 rs1281323 1.43 0.1514 rs1281338 0.68 0.1825 0.9775 rs2254327 1.20 0.4666 rs2254702 0.77 0.3262 0.3934 rs10911659 0.67 0.1191 rs234122 1.22 0.4752 0.2542 rs234092 0.88 0.6704 rs4650678 0.69 0.3239 0.2998 rs234095 0.74 0.3455 rs234122 1.22 0.4752 0.7135 rs6684195 1.07 0.7890 rs12030554 0.95 0.8735 0.5315 rs1200610 1.33 0.3384 rs7413268 NA NA 0.5710 rs1208517 1.57 0.1727 rs6424975 1.16 0.5814 0.2907 rs10489486 1.17 0.5726 rs6689206 1.20 0.4825 0.5581 rs2186024 0.91 0.7059 rs12119534 0.87 0.6666 0.6530 rs2494354 1.38 0.6329 rs2494155 NA NA 0.6841 rs2494312 1.53 0.5164 rs2494315 NA NA 0.6424 rs927724 1.38 0.3749 rs7518775 0.66 0.3943 0.1977 rs599779 0.88 0.6135 rs556744 1.13 0.7071 0.6567 rs1898240 0.94 0.8383 rs16844836 1.10 0.7876 0.6840 rs487359 0.65 0.0974 rs590448 0.88 0.6975 0.0696 rs1890133 1.45 0.3036 rs7538527 0.95 0.8832 0.3837 rs1400875 0.93 0.7980 rs2644112 1.40 0.2747 0.4743 rs2820312 0.97 0.9146 rs2820312 1.13 0.7156 0.6901 rs2050935 0.83 0.5526 rs12406229 0.71 0.3761 0.0653 rs4950978 0.77 0.3270 rs3862948 0.71 0.3380 0.5629 rs1470637 0.75 0.2661 rs9787334 0.71 0.2640 0.6768 rs8231114 0.86 0.0414 rs821114 0.42 0.0770 4.64E−06 rs280221 0.63 0.0947 rs966256 1.53 0.1499 0.7680 rs643930 1.52 0.1195 rs658347 1.07 0.7857 0.6112 rs10779481 0.85 0.7714 rs11119076 NA NA 0.8277 rs11119078 1.24 0.7355 rs11119079 NA NA 0.5064 rs1933564 0.83 0.4452 rs12043779 1.05 0.8821 0.6460 rs11119439 1.15 0.5851 rs11119426 1.08 0.8554 0.9201 rs590152 1.15 0.5851 rs845451 1.29 0.3782 0.6529 rs12124008 1.46 0.1492 rs11580728 0.88 0.7044 0.3009 rs3104209 1.77 0.0290 rs3104212 0.69 0.2494 0.0807 rs487208 1.24 0.4067 rs1890007 0.77 0.4210 0.1485 rs7548730 1.64 0.0655 rs7518358 1.09 0.7963 0.1064 rs7547186 1.94 0.0244 rs11572775 1.47 0.2693 0.0387 rs10495064 0.79 0.6309 rs10495065 1.10 0.7549 0.1417 rs6673733 0.62 0.0705 rs17046838 1.41 0.2314 0.1779 rs2377781 1.71 0.0428 rs10863375 1.29 0.3272 0.0148 rs9441857 0.73 0.2379 rs4846353 0.96 0.8905 0.3927 rs1341331 0.60 0.0442 rs16845973 0.76 0.3276 0.1993 rs750426 0.67 0.2191 rs750426 1.01 0.9805 0.3754 rs898833 0.67 0.2344 rs750426 1.01 0.9805 0.3946 rs11122571 1.37 0.2126 rs2296800 0.98 0.9581 0.6134 rs7540252 0.88 0.6022 rs2282319 0.91 0.7398 0.4219 rs1316408 1.30 0.3477 rs12082061 1.03 0.9301 0.3272 rs7542797 0.77 0.4822 rs10864669 0.93 0.7780 0.7962 rs487770 0.64 0.3261 rs607368 1.40 0.3102 0.3642 rs12746334 0.61 0.2846 rs11577962 NA NA 0.4971 rs12135445 0.84 0.6462 rs16838380 0.85 0.6353 0.4670 rs879081 1.12 0.6495 rs4659570 1.02 0.9501 0.2301 rs9287247 0.69 0.1627 rs882869 0.99 0.9792 0.1152 rs2066380 0.80 0.3605 rs11802581 1.22 0.5513 0.1461 rs9661248 1.10 0.7731 rs4658608 1.49 0.3460 0.3002 rs4654274 1.02 0.9341 rs2184975 0.84 0.5095 0.8109

Taken together, these data strongly support a genetic interaction between LRRK2 and PARK16 that initially impacts human CNS tissue physiology, as reflected by the transcriptome signature in unaffected carriers, and ultimately favors PD pathology in a small subset of individuals at risk.

Evidence of a LRRK2-RAB7L1 Pathway

As 5 candidate genes are present within the PARK16 locus (SLC45A3, NUCKS, RAB7L1, SLC41A1, and PM20D1), each of the genes were experimentally screened for a functional interaction with LRRK2 (FIG. 2A). A previously-described primary rat neuron in vitro culture model was used, in which transient expression of familial PD-associated LRRK2 G2019S or R1441C mutant alleles leads to a marked reduction in neurite process length (MacLeod et al., 2006). Overexpression of RAB7L1, but not other genes in the PARK16 locus, significantly suppressed the LRRK2 mutation-induced neurite length phenotype (FIG. 2B). RAB7L1 did not modify neurite length in the context of overexpression of wild-type LRRK2 (FIG. 2A). RAB7L1 is a small cytosolic GTPase, structurally distinct from RAB7 despite its name (also known as RAB29) (Shimizu et al., 1997). One of ˜60 small GTPases in the human genome, RAB7L1 has previously been shown to localize primarily to the Golgi apparatus and implicated in vesicular sorting in the context of Salmonella or Hepatitis C infection (Berger et al., 2009; Spano et al., 2011). But the function of RAB7L1 in CNS neurons remains unknown. Orthologues of RAB7L1 in other organisms, including C. elegans Glo-1 and Drosophila melanogaster Lightoid, have been implicated in trafficking to lysosome-related organelles (Hermann et al., 2005) and in the regulation of neurite process length (Grill et al., 2007), reminiscent of LRRK2 mutant phenotypes (MacLeod et al., 2006). Thus this gene was of particular interest.

Because GTPases such as RAB7L1 are typically only active in the GTP-bound state, mutant forms were generated that are constitutively active (CA; Q67L; this mutation is deficient in GTPase activity) or inactive (IN; T21N; a mutation within the presumptive GTP binding site). As expected, overexpression of the CA RAB7L1, but not IN RAB7L1, significantly suppressed the LRRK2 mutation-induced neurite length phenotype. Of other Rab family members, expression of RAB3A or RAB5A failed to rescue the phenotype, whereas RAB7 CA was effective in suppressing the process length shortening induced by LRRK2 mutation (FIG. 2B). In contrast to RAB7L1 overexpression, knockdown of RAB7L1 alone led to a significant reduction in neurite process length, similar to the effect of the LRRK2 G2019S mutant expression (FIG. 2B, 9B).

Next more direct evidence of a physical interaction between LRRK2 and RAB7L1 was sought and thus co-immunoprecipitation studies were performed. Epitope-tagged forms of LRRK2 and RAB7L1 (3×Flag-LRRK2 and GFP-RAB7 L1) were co-transfected into HEK293T cells, and after 48 hrs, cell lysates were immunoprecipitated with an anti-Flag antibody and then probed for RAB7L1. Flag-immunoprecipitation of LRRK2 effectively co-precipitated RAB7L1 (FIG. 3A). The interaction did not appear to be altered by the presence of the G2019S mutant, or using a kinase-dead variant K1906M of LRRK2 (MacLeod et al., 2006). Similarly, immunoprecipitation of RAB7L1 with an antibody to the GFP tag co-precipitated LRRK2 only in the presence of RAB7L1-GFP (FIG. 3B). To probe for an interaction between LRRK2 and RAB7L1 in a more physiological context, RAB7L1 protein was examined in brain lysates from transgenic mice that harbor human wild-type LRRK2 or a familial PD mutant form of LRRK2, R1441C, within a large bacterial artificial chromosome (BAC) construct. Transgenic LRRK2 is broadly expressed throughout the CNS of these mice, although at relatively low levels (FIG. 10A). Brain tissue lysates were immunoprecipitated for LRRK2 protein with a rabbit monoclonal antibody. Western blotting of the lysates for RAB7L1 demonstrated co-immunoprecipitation of RAB7L1 (FIG. 3C).

In vitro fluorescence microscopy studies were consistent with the presence of RAB7L1 and LRRK2 in common subcellular compartments. GFP-tagged RAB7L1, transfected into SH-SY5Y cells, localized primarily to the Golgi apparatus (as identified with the Golph4 marker), as well as along tubular structures emerging from Golgi apparatus, consistent with prior reports (Spano et al., 2011). LRRK2 staining appeared more diffuse than RAB7L1, but there was significant overlap (FIG. 3D). In contrast to the wild-type form, the RAB7L1 CA or IN mutant forms appeared more diffusely localized through the cytoplasm, as did a RAB7L1 alternative transcript (AT) deficient in the predicted GTP-binding region (FIG. 3D); accumulation of the IN and AT mutant proteins was significantly reduced (FIGS. 3D and 10B).

In Vivo Analysis of a LRRK2-RAB7L1 Pathway in Drosophila Dopamine Neurons

To pursue potential mechanisms of LRRK2 pathology in vivo, a Drosophila model was established. Although transgenic mouse models expressing mutant LRRK2 have been widely described (Andres-Mateos et al., 2009; Li et al., 2009; Piccoli et al., 2011; Tong et al., 2009), these do not show consistent neurodegenerative phenotypes. Dopamine neuron-selective expression of human familial PD-associated G2019S-mutant LRRK2—using either a tyrosine hydroxylase (TB) (Friggi-Grelin et al., 2003) or dopa decarboxylase (DDC) promoter-Gal4 driver (Fischer et al., 1988)—induced premature mortality of young adult animals (FIG. 4A; nontransgenic mean lifespan 37.1 days+/−1; G2019S mean lifespan 4.8 days+/−0.2), akin to previous reports (Ng et al., 2009). In contrast, transgenic expression of wild-type human LRRK2 did not lead to a discernible phenotype. Furthermore, expression of the mutant G2019S LRRK2 transgene in several other cell types, including motor neurons, eye tissues, or muscles (using a variety of promoter-Gal4 driver constructs), failed to lead to a discernible effect on survival or otherwise.

Subsequently a targeted screen for potential genetic modifiers of the LRRK2 G2019S mutant phenotype was performed, based on the idea that LRRK2 may modify a specific intracellular trafficking process, and focused on RAB7L1. A series of 16 Drosophila Rab genes, (see Table 4; out of 33 identified in Drosophila), or CA or IN forms of these (Zhang et al., 2007), were investigated.

TABLE 4 Rab GTPase genes screen for a rescue of the LRRK2 G2019S phenotype in Drosophila. Average adult TH-driven Transgene lifespan Rab GTPase mutation (days) SEM n Rab1CA Q70 5.9 0.43 23 Rab2CA Q65 4.7 0.44 27 Rab3CA Q80 7 0.5 22 Rab4CA Q67 6 0.36 21 Rab5CA Q88 5.9 0.37 20 Rab6CA Q71 5.6 0.44 22 Rab7WT n/a 10.2 0.59 21 Rab7L1 DN T33 5.6 0.53 22 Rab7L1WT n/a 23.3 1.09 52 Rab7L1CA Q79 24 1.11 45 Rab8CA Q67 6.8 0.39 21 Rab9CA Q71 5.3 0.4 23 Rab10WT n/a 5.6 0.39 22 Rab14CA Q94 6.6 0.37 20 Rab18CA A64 4.6 0.42 20 Rab23CA Q96 6.7 0.49 20 RabX2CA D66 4.8 0.4 20 RabX4CA Q67 5.9 0.52 22

Briefly, LRRK2 G2019S mutants were mated with a panel of previously described transgenic Drosophila strains that allow for overexpression of wild-type (WT) or constitutively active (CA), forms of the Rab genes (Zhang et al., 2007), using a standard balancer chromosome-based mating scheme. Co-expression of a majority of these Rab transgenes with LRRK2 within dopamine neurons produced no effect on the survival of animals co-expressing LRRK2 G2019S (FIG. 4A; Table 4). In contrast, overexpression of wild-type and CA forms of the Drosophila RAB7L1 orthologue (termed lightoid) afforded a dramatic rescue of the LRRK2 G2019S premature mortality phenotype (mean lifespan 24.0 days+/−1 for the CA; FIG. 4A). Of note, among the other Rabs screened, only Rab7 led to a statistically significant—albeit much weaker—survival benefit (mean lifespan 14.3 days+/−0.6). Rab1, which was previously found to rescue a defect in vesicular trafficking to the Golgi apparatus in alpha-Synuclein overexpression models of PD (Cooper et al., 2006), did not rescue the LRRK2 defect, suggesting distinct mechanisms.

Next, dopamine neuron survival at the dorsomedial posterior protocerebral (PPM1) and dorsolateral posterior protocerebral (PPL1) clusters of Drosophila CNS mushroom bodies was quantified in terms of the loss of expression of a dopamine neuron-specific nuclear localization signal (NLS)-GFP marker protein, using fluorescent confocal microscopy analysis of whole mounted tissue. Expression of LRRK2 G2019S, but not the WT form, led to the preferential loss of neurons in the dorsomedial cluster, reminiscent of the phenotype in other Drosophila models of PD (Feany and Bender, 2000). Co-expression of CA RAB7L1 rescued the LRRK2 G2019S dopamine neuron loss phenotype (FIG. 4B). Deficiency of the RAB7L1 orthologue (in lightoid homozygous mutants) selectively in dopamine neurons by expression of an siRNA construct (Dietzl et al., 2007), led to a significant loss of dopamine neurons (FIG. 4B).

PARK16 Risk Variants Modify RAB7L1 Splicing and Expression

The combination of human brain transcriptomic, human genetic, and model system studies support a role for PARK16, and specifically the PARK16 locus gene RAB7L1, in a pathway with LRRK2. Next possible molecular mechanisms at play at the PARK16 locus that may be responsible for a link between common genetic variants, RAB7L1 function, and PD risk were investigated. A challenge to this is that typically many variants at a given chromosomal location are so closely associated (in ‘linkage dysequilibrium’) so as to make impossible the identification of which is truly ‘causal’ rather than just coincidental. On reanalysis of existing genome-wide splicing data from human lymphoblasts (Montgomery et al., 2010), the PD-associated PARK16 haplotype was found to be associated with alternative splicing of RAB7L1, characterized by the skipping of exons 2 and 3. It is noted that a common SNP variant within the PARK16 locus, rs1572931, that is in linkage dysequilibrium with SNP rs947211 (Hamza et al., 2010) and thus similarly linked to PD risk, falls precisely within regulatory sequences for splicing at the Intron1-exon2 boundary (FIG. 5A). Akin to the lymphoblast transcriptome splicing data, analysis of a set of human cortical brain samples revealed that the rs1572931 genotype is similarly associated with modified splicing of RAB7L1 in human forebrain (FIGS. 5B, 12A; see Table 6), where the protective PARK16 haplotype is associated with increased exon 2 inclusion in RAB7L1 mRNA. Based strictly on human gene expression data, a causal role for SNP rs1572931 in altered splicing of RAB7L1 cannot be directly assigned (as other SNPs in linkage disequilibrium could be responsible for the observed association). Thus the causal effect of rs1572931 was evaluated using minigene reporter vectors that harbor either the risk-associated or protective allele at rs1572931, but are otherwise identical (FIGS. 5Cii, 12B). Upon transfection into SH-SY5Y human neuroblastoma cells, the rs1572931 risk allele led to increased RAB7L1 exon 2 skipping relative to the protective allele (FIGS. 5D, 12C-E).

TABLE 6 Human Brain Cortical Samples. Brain ID Age Sex Status rs1572931 B1 80 F Unaff. AA B2 82 M LBD AA B3 56 F ALS GA B4 62 F ALS GA B5 67 M ALS GA B6 72 M Unaff. GA B7 87 M Unaff. GA B8 57 F Unaff. GA B9 84 M Unaff. GA B10 58 M FTD GA MND B11 85 M FTD GA MND B12 87 F PD GA B13 84 M PD/D GA B14 80 M PD/D GA B15 68 F PSP GA B16 83 M AD GG B17 89 F AD GG B18 79 F AD GG B19 89 F AD GG B20 89 F AD GG B21 83 M AD GG B22 73 M AD GG B23 86 F AD GG B24 75 F AD GG B25 62 F AD GG B26 89 F AD GG B27 89 F AD GG B28 89 M AD GG B29 88 M AD GG B30 76 M AD GG B31 80 M ALS GG B32 64 M ALS GG B33 60 M ALS GG B34 79 F ALS GG B35 66 M ALS GG B36 88 F ALS GG B37 71 F ALS GG B38 76 F Unaff. GG B39 57 M Unaff. GG B40 80 M Unaff. GG B41 65 F Unaff. GG B42 62 M Unaff. GG B43 89 M Unaff. GG B44 89 M Unaff. GG B45 57 F Unaff. GG B46 52 F Unaff. GG B47 80 M FTD GG B48 61 M FTD GG B49 74 M FTD GG B50 50 M FTD GG MND B51 77 M FTD GG B52 73 M FTD GG B53 84 M LBD GG B54 80 M LBD GG B55 76 F PD GG B56 83 M PD GG B57 74 M PD GG B58 80 F PD GG B59 77 F PD GG B60 80 F PD GG B61 85 M PD GG B62 77 M PD GG B63 77 F PD/D GG B64 84 F PD/D GG B65 81 F PD/D GG B66 69 F PD/D GG B67 65 M PD/D GG B68 72 F PD GG

Exon skipping is predicted to lead to the formation of a truncated form of RAB7L1 protein that lacks the predicted GTP-binding domain in the amino-terminal region (FIG. 12C). Overexpression of this truncated form leads to low level accumulation of a shortened protein product (FIG. 10B), and reduced localization to the Golgi apparatus (FIG. 3D); although the shortened product can bind with LRRK2 protein (FIG. 3B), expression of this truncation mutant in primary neurons failed to rescue the reduced neurite length phenotype associated with G2019S mutant LRRK2 (FIG. 12F), whereas expression of the wild-type RAB7L1 effectively rescued the phenotype. Consistent with these in vitro findings, a significant reduction in full-length RAB7L1 protein was observed in cerebral cortex tissue from unaffected individuals who carry the PARK16 risk allele, when compared to non-carrier individuals (FIG. 5E). It is noted that a similar reduction is seen in PD patient cerebral cortex tissue regardless of the PARK16 genotype. This appears specific to PD, as no such decrease is observed in tissue from patients suffering from other neurodegenerative disorders examined (frontotemporal dementia or amyotrophic lateral sclerosis) who do not carry the PARK16 risk allele (FIG. 5E). Taken together, these findings argue in favor of a post-transcriptional (splicing) mechanism of action for the PARK16 PD risk variant's impact on RAB7L1 levels. However, given the linkage disequilibrium structure of the region, additional transcriptional regulatory effects may exist (Gan-Or et al., 2012).

Lysosomal Changes and Retromer-Associated Sorting Defects in LRRK2 and RAB7L1 Mutant Neurons

A cellular role for the LRRK2-RAB7L1 pathway was investigated. Prior studies have broadly implicated both of these gene products in intracellular sorting (Sakaguchi-Nakashima et al., 2007; Spano et al., 2011). Expression of the LRRK2 G2019S clinical mutation in rat primary neurons induced lysosomal swelling, as quantified by immunostaining for the lysosomal marker LAMP2 or using the lysosomotropic dye Lysotracker, consistent with prior work and other studies (Dodson et al., 2012; MacLeod et al., 2006; Stafa et al., 2012) (FIG. 6A). In addition to lysosomal enlargement, there was significant reduction in lysosomal accumulation of the cation-independent mannose 6-phosphate receptor (MPR) in terms of the fraction of LAMP2-positive structures stained with MPR. As MPR is required also for the recruitment of lysosomal hydrolases, its deficiency is predicted to lead to functional lysosomal deficits. Knockdown of RAB7L1 was similarly associated with swollen lysosomes and reduced lysosomal MPR, whereas overexpression of RAB7L1 suppressed the lysosomal phenotypes in the context of LRRK2 G2019S expression (FIG. 6A).

MPR is typically recycled between the endolysosome compartment and the Golgi apparatus by the retromer complex (Arighi et al., 2004; Bonifacino and Hurley, 2008; Seaman, 2009; St. George-Hyslop et al., 2009). Given the primary apparent localization of RAB7L1 to the Golgi apparatus (FIG. 3D), as well as the enrichment of LRRK2 at this organelle (FIG. 3D)(Stafa et al., 2012), without being bound by theory, the lysosomal compartment defects described above may be secondary to altered retromer mediated trafficking machinery between these organelles (Bonifacino and Hurley, 2008; Seaman, 2004). Analysis of Golgi structures by immunostaining with the Golph4 marker in primary neurons transfected with either LRRK2 G2019S or shRNA for RAB7L1 did not reveal evidence of gross structural changes, but MPR co-localization at the Golph4-positive Golgi apparatus structures was significantly reduced (FIG. 6B). Accumulation of MPR at early endosomes, assessed by co-staining with the marker early endosomal antigen-1 (EEA1; FIG. 6C), did not appear altered, whereas accumulation at the cell surface appeared increased. The total areas of Golph4, MPR, or EEA1 staining were unaffected by G2019S LRRK2 expression or RAB7L1 knockdown (FIGS. 6A-C).

The retromer complex is required for retrograde transport of selective cargo—including MPR—between lysosomes and the Golgi apparatus, through endosomal intermediates, in mammalian cells (FIG. 6D) (Bonifacino and Hurley, 2008; St. George-Hyslop et al., 2009), and defects can lead to lysosomal swelling (Arighi et al., 2004). Furthermore, rare mutations in a retromer component, VPS35, were recently linked to rare familial forms of PD (Vilarino-Guell et al., 2011; Zimprich et al., 2011). Knockdown of VPS35 in primary neuron cultures led to reduced MPR co-localization with the Golgi apparatus and with late endosomes/lysosome markers (FIGS. 6A-B), as previously described (Seaman, 2009). Similarly, expression of a familial PD-associated mutation in VPS35, D620N (Vilarino-Guell et al., 2011; Zimprich et al., 2011), phenocopied the MPR missorting phenotype of G2019S mutant LRRK2 expression or VPS35 knockdown (FIGS. 6A-B), suggesting a dominant negative mode of action which is consistent with a predicted structural alteration of a retromer complex interaction motif (Vilarino-Guell et al., 2011; Zimprich et al., 2011). In contrast, overexpression of wild-type VPS35, which promotes trafficking through the retromer pathway, suppressed the altered MPR localization seen with G2019S mutant LRRK2 expression (FIGS. 6A-B). Thus, although it is likely that the LRRK2-RAB7L1 pathway impacts intracellular sorting processes in addition to retromer complex function, suppression of retromer dysfunction is sufficient to rescue the deficits associated with defects in the LRRK2-RAB7L1 pathway.

The functional relationship of VPS35 with the LRRK2-RAB7L1 pathway was further investigated in the context of neurite process maintenance. In rat primary neurons, overexpression of VPS35 alone did not directly modify neurite process length, but effectively suppressed the loss of neurite processes in the context of LRRK2 G2019S expression or RAB7L1 knockdown (FIG. 7A). In contrast, knockdown of VPS35 with an shRNA vector, or expression of the VPS35 D620N mutant form, led to neurite process length reduction that phenocopied the effect of LRRK2 G2019S expression. In vivo analysis in the Drosophila CNS further supported a role for retromer dysfunction in the context of LRRK2-RAB7L1 pathway defects. Overexpression of Drosophila VPS35 in Drosophila CNS dopamine neurons rescued the LRRK2 G2019S dopamine neuron loss phenotype (FIG. 7B), and similarly extended the lifespan of G2019S LRRK2 mutant-expressing flies. In contrast, knockdown of VPS35 selectively in Drosophila TH-positive dopamine neurons led to significant cell loss and a reduced lifespan (FIG. 7B).

Reduction in Retromer Complex Component Levels in the Context of LRRK2-RAB7L1 Pathway Defects.

Next potential molecular mechanisms for the apparent defects in retromer pathway function in the context of LRRK2 G2019S mutation or RAB7L1 knockdown were investigated. In mouse N2A neuroblastoma cells, expression of LRRK2 G2019S or knockdown of RAB7L1 led to a significant reduction in the levels of accumulated VPS35 as well as VPS29, a second component of the retromer complex (FIG. 7C). Levels of retromer complex components are dependent on the formation of the entire complex, which also includes VPS29, and thus loss of any complex component is predicted to impact levels of others (Kim et al., 2010). Analysis of transgenic mouse total brain tissue overexpressing the R1441C mutant form of LRRK2 also led to a significant reduction in the accumulation of VPS35 and VPS29, and VPS26 (FIG. 7D).

Although the precise mechanism by which the LRRK2-RAB7L1 pathway impacts retromer complex function and levels remains to be determined, co-immunoprecipitation studies of LRRK2 with VPS35 support a direct interaction between these proteins: Lysates from SH-SY5Y cells co-expressing epitope-tagged V5-LRRK2 (or vector) and eGFP-VPS35 forms, were immunoprecipitated for the eGFP tag. Subsequent Western blotting revealed co-purification of LRRK2 with eGFP-VPS35 (FIG. 7E). Similarly, immunoprecipitation of LRRK2 from LRRK2 transgenic mouse brain tissue led to the co-precipitation of endogenous VPS35 (FIG. 7F). It is possible the interactions of LRRK2 with VPS35 and RAB7L1 are within a single complex or multiple complexes.

To relate those findings to sporadic PD, VPS35 levels in PD or unaffected human brain tissue were analyzed. First a meta-analysis of substantia nigra (SN) mRNA expression levels in 5 publically available microarray gene expression datasets from patients and controls was carried out (Table 5; totally 144 individuals, 63 unaffected individuals and 81 PD patients), and a highly significant decreased in VPS35 mRNA levels (FIG. 7G) was observed.

TABLE 5 GEO datasets used for the meta-analysis of VPS35 mRNA levels in SN. Fold changes and p-values for each individual dataset are indicated. Re- Dis- Fold Dabaset Probe gion ease change n p-vaue GSE26927 ILMN_21093 SN PD −27% 20 4.90E−03 GSE8397 217727_x_at SN PD −10% 28 1.47E−01 GSE7621 217727_x_at SN PD −14% 25 1.18E−01 GSE20292 217727_x_at SN PD −29% 29 2.80E−04 GSE202923 217727_x_at SN PD −9% 26 3.40E−01 GSE20159 ILMN_1761721 SN LBD −29% 33 3.00E−02

Such a decrease was also observed in gene expression data from laser-microdissected PD SN dopamine neurons, when compared to similar cells isolated from unaffected patients (FIG. 7G), as well as in PD cerebral cortex tissue (FIGS. 7H-7I). Taken together, these results support a role for retromer deficiency in the impact of PD-associated genetic risk variants on brain neurons.

Discussion

Using a brain transcriptomic approach as a starting point, evidence is provided that the impacts of several distinct PD risk-associated common genetic variants are overlapping, even in unaffected PD-free carrier tissue. This points to a convergent pathway of action for such variants. Focusing subsequently on LRRK2 and the PARK16 locus gene RAB7L1, in vitro and in vivo studies support a functional interaction: these gene products bound together and functionally interacted in the regulation of neurite process length in vitro, as well as in the context of dopamine neuron survival in vivo. The impact of LRRK2 and PARK16 variants on brain gene expression was observed even in unaffected carriers of the PARK16 or LRRK2 locus risk variants suggesting the existence of a pre-disease prodromal state in such carriers, that favors subsequent progression.

The most prominent neuronal sorting phenotypes observed in the context of PD-associated LRRK2-RAB7L1 pathway changes were at lysosomes and the Golgi apparatus. Without being bound by theory, the proximal site of action for these proteins may be in defective retromer function at the Golgi apparatus, given the enrichment of both proteins at this structure. Trafficking of MPR to the Golgi apparatus—a function of the retromer complex—is defective, and associated with lysosomal swelling. Although the precise mechanism of retromer dysfunction is unclear, retromer pathway components including VPS35 appear reduced in the context of LRRK2 mutation or RAB711 knockdown. Recently described familial PD-associated clinical mutations in VPS35 phenocopy the deficits associated with LRRK2-RAB7L1 pathway dysfunction, whereas overexpression of VPS35 can rescue such deficits. It is also noted that RAB7 was identified in both the in vitro and in vivo screens of RAB proteins as suppressing the phenotype of LRRK2 mutant pathology, albeit less robustly than RAB7L1. RAB7 is the only RAB protein previously implicated in the regulation of retromer function (Rojas et al., 2008).

Prior studies have supported a role for LRRK2 in vesicular trafficking (Biskup et al., 2006; Dodson et al., 2012; Higashi et al., 2009; MacLeod et al., 2006; Stafa et al., 2012). However, cellular mechanisms of LRRK2 relevant in human brain—and in the context of PD or PD risk variants—have remained unclear. The studies herein are unusual in pursuing a PD genetic pathway using both human brain and model system analyses. A genetic interaction between LRRK2 and RAB7L1 was identified in the context of PD risk, and variants at the loci of these genes impact the brain transcriptome in an overlapping manner. Subsequent cell and animal model studies support a model where LRRK2 and RAB7L1 defects may modify intracellular sorting and retromer pathway function.

It is possible that PD-related defects in LRRK2 and RAB7L1 adversely impact aspects of vesicular trafficking unrelated to retromer function. Nonetheless, inducing retromer function appears sufficient to rescue cellular defects and neuronal survival in these models, suggesting a therapeutic venue in PD patients. It is interesting to note that VPS35 deficits, as well as genetic variants at retromer complex receptor loci such as SORLA (Rogaeva et al., 2007), have also been associated with a second major neurodegenerative disorder, Alzheimer's disease (Muhammad et al., 2008); this suggests a broader role for retromer dysfunction in neurodegeneration. Without being bound by theory, different cargos may be involved in the association of the retromer pathway with distinct pathological processes in Alzheimer's and Parkinson's. To this end, it is of interest to investigate the impact of such retromer dysfunction on aSyn and other proteins associated with PD pathology.

Experimental Procedure

Drosophila Methods

Drosophila were cultured by standard methods on yeast-cornmeal-agar medium at 25° C. Wild-type and mutant G2019S LRRK2 transgenes were expressed specifically in catecholaminergic neurons, including dopamine neurons, using the Gal4-UAS system described (Fischer et al., 1988). Driver lines used include OK6 (motor neuron), Gmr (eye), G14 (muscle), TH (dopaminergic neuron), and DDC (dopaminergic neuron). A UAS-GFP::nuclear localization sequence (NLS) marker was used to visualize nucleii of cells in which trangenes were expressed (stock 4775 (w1118; P{UAS-GFP.nls}14), Drosophila Stock Center, Bloomington, Ind.). For the RAB screen, UAS-LRRK2 (G2019S) transgenic Drosophila, crossed with the TH-Gal4 driver, were screened against a UAS-Rab transgenic library (Zhang et al., 2007). Crossings were typically performed using standard balancer chromosome techniques. To generate strains in which the homozygous LRRK2 transgene and another (Driver or marker) transgene lay on the same chromosome (III), genetic recombination was using standard techniques. Adult Drosophila mushroom bodies were dissected as in (Wu and Luo, 2006) and imaged immediately, without fixation, using a Zeiss LSM510 Meta confocal fluorescent microscope. For mortality curves, transgenic Drosophila surviving through adult metamorphosis were counted daily, from the day of pupal eclosion onward. Locomotion deficits were assessed by methods know in the art.

Primary Neurons Culture

Sprague-Dawley rat or mouse P1 primary dissociated cortical cultures were prepared and transfected essentially as described (Xia et al., 1996) with the following modifications: cells were plated at high density, approximately 250,000 cells/cm2, in 24-well plates with 500 ul medium/well. Culture medium used for plating cells was Neurobasal-A supplemented with 2% B-27 and 10% FBS. At 1 day after plating, medium was changed to reduced serum (1% FBS+ added antimitotic agents: 70 μM uridine and 25 μM 5-fluorodeoxyuridine) and replaced weekly thereafter; for transfections no DMSO was added to the transfection mixture, cells were not subjected to glycerol shock, and a total of 3 μg plasmid DNA was used per well. Cells were fixed in 4% PFA and immunostained with mouse α-RAB7L1 (Santa Cruz, 1:100), and rabbit monoclonal α-LRRK2 (Michal J. Fox Foundation MJFF4, 1:100), then with appropriate fluorescent secondaries (Jackson, 1:1000-2000). Neurite length and neurite puncta (defined as swellings greater than 2 um in diameter) were counted for at least 20 neurons per condition. Mean puncta number per neuron was normalized to total average neurite length versus wild-type LRRK2 transfected cells. Fluorescent microscopy was performed using a Nikon TE 2000-S microscope and a Zeiss LSM510 Meta confocal microscope. Images were analyzed using Image-Pro Plus (Mediacybernetics) software version 5.1.0.20.

Colocalization Analysis

Primary rat cortical neurons were cultured on glass coverslips, transfected, and fixed as previously described in this methods section. Cells were immunostained for MRP (Abcam #ab2733, 1:400), Golph4 (Abcamab #28049, 1:500), Lamp2 (Sigma L0668, 1:500). Fluorescent microscopy was performed using a Zeiss LSM510 Meta confocal microscope. Images were analyzed using NIH ImageJ software version 1.45.

Cell Culture, Transfection and Cytochemistry

HEK293T and SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37° C. in a 5% CO₂ atmosphere. Transient expression was performed by transfecting the plasmids using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. The transfected SH-SY5Y cells grown on glass coverslips for 24 hours were fixed with 4% paraformaldehyde in PBS for 30 minutes, washed three times with PBS and subjected to the observation of fluorescence. For immunostaining of golgi, fixed cells were blocked and permeabilized with PBS containing 0.1% TritonX-100 and 3% bovine serum albumin followed by incubation with anti-Golph4 polyclonal antibody (abcam) and Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen). Staining of nuclei was performed by using SYTOX Orange nucleic acid stain (Invitrogen). Fluorescence was detected using Zeiss LSM 510 confocal microscope.

Immunohistochemistry and Signal Quantification

LRRK2 R1441C or Wt BAC transgenic mice (Li et al., 2009) (Jackson Laboratory) were sacrificed and perfused immediately with 4% PFA for 20 min. Brains were cut by vibratome into sections 60 μm thick. Sections were blocked in 5% NDS overnight at 4 C, then incubated with primary antibodies overnight at 4 C. Antibodies used were sheep monoclonal α-TH (Pelfreeze, 1:500), mouse α-RAB7L1 (Santa Cruz, 1:100), and rabbit monoclonal α-LRRK2 (Michal J. Fox Foundation MJFF4, 1:100). Sections were incubated at room temperature for 2 hours with appropriate fluorescent secondaries (Jackson Laboratories, 1:1000). Microscopy was performed with a Zeiss LSM510 Meta confocal. Fluorescence signal intensity was quantified using NIH ImageJ.

Human Autopsied Brain Samples

Cortical BA9 area brain samples were obtained from the New York Brain Bank and are detailed in Table 5. Anonymous, de-identified tissue from the brain bank was used.

Quantitative Real Time RT-PCR

RT-qPCR was done as described in (Rhinn et al., 2008) RAB7L1 ratio was quantified using ΔΔCt method using primers pairs: RAB7L1_Ex2_fw (CAGCAAACACTACAAGTCCACG) (SEQ ID NO:15) and RAB7L1_Ex3_rv (CAGCTGAAGCCGCACTATCTCG) (SEQ ID NO: 16); RAB7L1_Ex4_fw (GACAGCAAGCTCACACTACCCA) (SEQ ID NO:17); RAB7L1_Ex5_rv (TCTGTCCAACCTGTGAAACCGT) (SEQ ID NO:18) for human brain samples.

Minigene Splicing Assay

The human SH-SY5Y neuroblastoma cell line (ATCC) was cultured following ATCC's instructions, plated at densities of 4.10e5 cells per well (48-well plates) in wells coated with 0.1% gelatin (Specialty Media, Millipore) 24 hours prior to transfections. Transfections were performed with Lipofectamine 2000 reagent (Invitrogen) following manufacturer's instructions. After transfection with plasmids encoding the reporter construct, RNA was extracted using miRNeasy kit (Qiagen) and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) following manufacturer's instructions. The cDNA was amplified by PCR using the following primers: GGAGGGCGTCTAGGGAATCGAG (SEQ ID NO: 19) (Fw, complementary to exon1 of RAB7L1) and CTTCAGGGTCAGCTTGCCGTAG (SEQ ID NO: 20) (Rev., complementary to GFP CDS) and Accuprime high-fidelity polymerase (Invitrogen) following manufacturer's instruction with an hybridization at 55 C and an elongation step of 1 min. Pictures from an ethidium bromide stained agarose gels of the migrated PCR products was analyzed using ImageJ software.

Supplementary Experimental Procedures

Western Blots

Mouse brain protein fractions were prepared as follows. Mouse striata were dissected and homogenized by motorized dounce in Krebs-Ringer buffer with 0.32 M sucrose, then centrifuged at 3000×g for 10 min. Supernatant was collected and centrifuged at 10,000×g for 30 min. Pellet was resuspended in NuPage loading buffer (Invitrogen). Human brain proteins were prepared from frozen blocks using RIPA reagent (Pierce) following manufacturer's instruction. SDS-Page and Western Blot were performed according to manufacturer's protocols with NuPage Bis-Tris Mini Gel and Xcell II Blot Module (Invitrogen). Antibodies used include: LRRK2 (MJFF #1 & #2, 1:200), Rab5 (Abcam ab18211, 1:500), RAB7L1 (clone 2B8, Sigma, 1:400, clone 31-E, Santa Cruz sc-81924, 1:400), anti-Flag M2 (Sigma, 1:1000), anti-GFP (Covance, 1:1000), anti-alpha-tubulin (DM1A, 1:2000), SNAP25 (Abcam ab41455, 1:500), VAMP2 (Abcam ab3347, 1:500), beta-actin (clone C4) (Abcam ab3280, 1:800) and appropriate HRP-conjugated secondaries (Jackson, 1:2000). Blots were visualized using Supersignal luminol substrate (ThermoScientific #34075).

Immunoprecipitation

For cultured cells, HEK293T cells transfected for 48 hours were lysed with lysis buffer containing 0.5% Triton-X, 1 mM EDTA and protease inhibitor cocktail (Sigma). The lysates were rotated at 4° C. for 1 hr followed by centrifugation at 20,000 g for 5 min. The supernatant was added to 30 ul (slurry volume) of Dynabeads protein G (Invitrogen) preincubated without (preclear) and with an anti-flag M2 monoclonal antibody (Sigma) and the mixture was rotated for 30 min at 4° C. The beads were washed three times with ice-cold PBS and subjected to immunoblotting.

For mouse tissue, whole brains were dissected and homogenized by motorized dounce at 0° C. in Invitrosol detergent (Invitrogen) and 1× Protease inhibitor cocktail (Pierce) according to manufacturer's protocol Lysate was incubated shaking for 30 min at 4° C. with gel beads (Pierce Co-IP kit #26149) covalently conjugated to LRRK2 antibody (either MJFF #2 or #4) or a control IgG antibody. Beads were washed 4×10 min each, and then eluted. Eluant was analyzed by Western Blot, probed for LRRK2 (MJFF #1, 1:200), Rab 7L1 (Santa Cruz sc-81924, 1:400), Rab11 (Abcam ab3612, 1:400), and beta-actin (clone C4) (Abcamab3280, 1:600).

DNA Constructs

The plasmid encoding rat RAB7L1 cDNA sequence was purchased from Open Biosystems, and the sequence was digested and ligated into BglII-EcoRI site of pEGFP-C1 expression vector (Clontech) to generate N-terminally GFP-tagged RAB7L1. As the purchased RAB7L1 sequence contained 286 bp insertion in the middle of cDNA resulting in the generation of stop codon, this insertion was removed by a long-PCR protocol. The plasmids encoding constitutive active (Q67L) and dominant negative (T21N) rat RAB7L1 were generated by using site-directed PCR-mutagenesis kit (Stratagene) from the plasmid encoding N-terminally GFP-tagged wild-type RAB7L. All sequences were verified by DNA sequencing. Plasmids encoding wild-type and mutant Rab7 constructs were from Addgene; Rab3 and Rab5 constructs were also used. Plasmids encoding full-length human LRRK2 (wild-type, G2019S, K1906M) tagged with 3×FLAG at the N-terminus were used. Splice reporter minigene bearing plasmid was created by insertion of a synthesized sequence corresponding to the first exon, the first intron and the second exon and 200 bp of the second intron of human RAB7L1 gene in a pEGFP-N1 vector (Clontech) between its XhoI and HindIII restriction sites. Rab7L1 shRNA plasmid came from Sigma (MISSION shRNA clone NM_144875). LRRK2 plasmids used were those published (MacLeod et al., 2006), and confirmed.

PolyA-RNAseq

Library generation and sequencing: First-strand cDNA was synthesized from 1 μg of RNA per biological sample using SuperScript III (Invitrogen) following manufacturer's instructions and using the pdT-FS oligonucleotide to prime the reverse transcription. Barcoded first-strand samples from different samples were then pooled and treated with RNase H (Invitrogen) at 37° C. for 20 minutes followed by 15 minutes at 75° C. to degrade RNA template. First-stand cDNA was then purified using QIAquick PCR Purification kit (Qiagen) in a total volume of 30 uL. Second-strand cDNA was synthesized from 25 uL of first-strand cDNA template by adding 10 μl 10× buffer 2 (NEB), 5 μl 10 mM dNTPs, 20 U Klenow Fragment (3′→5′ exo-; NEB), 10 μl of 100 μM tagged 2nd strand primer (R-SS oligonucleotide: 5′-TCCGATCTGA-3′ with N=A,C,T,G mix (SEQ ID NO: 21)) and 46 μl water. The reaction mix was incubated at 37° C. for 30 minutes, followed by 10 minutes at 75° C. then cooled down at 4° C. Double-stranded cDNA was purified using PureLink PCR micro columns (Invitrogen) in a 30 uL volume. Illumina-compatible libraries were then generated by PCR from 25 uL of double-stranded cDNA template using Accuprime Pfx polymerase (Invitrogen) following manufacturer's instruction with NNSR forward (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGA TCTCT-3′(SEQ ID NO: 22)) and NNSR reverse (5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTC CGATCTGA-3′ (SEQ ID NO: 23)) primers. Thermo-cycling conditions were 2 min at 94° C. followed by 2 cycles of 94° C. for 10 s, 40° C. for 2 min, 68° C. for 1 min; 8 cycles of 94° C. for 10 s, 60° C. for 30 s, 68° C. for 1 min; 15 cycles of 94° C. for 15 s, 60° C. for 30 s, 68° C. for 1 min with an additional 10 s added at each cycle; and 68° C. for 5 min before cooling to 4° C. Amplified libraries were purified using PureLink PCR micro columns (Invitrogen) and directly used to generate clusters for sequencing-by-synthesis using the Illumina HiSeq 2000 platform. 100 bp single-end reads were obtained by sequencing to generate more than 300 million reads for the 34 samples.

Data was analyzed using Galaxy (Goecks et al., 2010): Illumina reads were converted to FASTQ Sanger format using FASTQ Groomer, the first 27 bp at their 5′ends of the reads were trimmed using FASTQ Trimmer to remove the polyA and adapters sequences and mapped to human hg19 genome using Burrows-Wheeler Alignment tools (Langmead et al., 2009) with Galaxy's default settings allowing 4% of missing alignments. All those tools are included in the Galaxy NGS toolset.

Human Sample Genotyping

DNA was extracted from brain samples using DNeasy kit (Qiagen) and amplified by PCR using primers RAB7L1_Genot_fw (GGTGAGCCTCCGCACTCG) (SEQ ID NO: 24) and RAB7L1_Genot_rv (TTCCCACCCACCGCCTGT) (SEQ ID NO: 25) and Accuprime polymerase (Invitrogen) following manufacturer's instruction with an hybridization at 55° C. and an elongation step of 1 min. PCR products were purified using PureLink PCR columns (Invitrogen) submitted to Sanger sequencing (GeneWiz, NJ) using RAB7L1_Genot_fw primer and analyzed using SeqScanner (Applied Biosystems).

GWAS Epistasis Analysis

The AJ GWAS dataset included a total of 278 cases and 178 controls that were genotyped using the Illumina Human 610-quad bead arrays (Cases n=91 and controls n=96) or the Illumina Human 660-quad bead arrays (Cases n=191 and controls n=84). Details of the genotyping and quality control assessments are provided in Liu et al (2011). Subjects were participants in two studies the Genetic Epidemiology of Parkinson Disease and the AJ study. Ascertainment and a description of the study participants is provided in detail in Marder et al (Marder et al., 2003) and Liu et (Liu et al., 2011). All subjects in the GWAS AJ dataset were genotyped for the LRRK2 G2019S mutation and for GBA mutations common in the AJ population (N3705, L444P, 84insGG, IVS2+1 G>A, V394L, R496H, D409H, A456P and V460V). For the epistasis analysis LRRK2 and GBA mutation carriers were removed from the dataset. The final dataset is comprised of 239 PD cases and 178 controls.

In addition to the AJ dataset, 3 additional publicly available GWAS datasets were studied and downloaded from NCBI's dbGap repository (Mailman et al., 2007): NGRC (CIDR/NGRC Genes and Environment, dbGap phs000196.v2.p1, (Hamza et al., 2010)), that comprises 2013 cases and 1995 controls, NINDS (NINDS-Genome-Wide Genotyping in Parkinson's Disease, dbGap phs000089.v1.p1, (Fung et al., 2006)) that comprises 267 cases and 270 controls and Mayo (Mayo-Perlegen LEAPS (Linked Efforts to Accelerate Parkinson's Solutions) Collaboration, dbGap phs000048.v1.p1, (Maraganore et al., 2005)) that comprises 443 cases and 443 controls. All subsequent genetic analysis were done using gplink (Purcell et al., 2007) software. Epistasis between PARK16 (rs823114, as identified to modify PD age of onset in LRRK2 mutations carriers (Latourelle et al., 2011)) and LRRK2 (rs11176052 as identified by PD GWAS meta-analysis (Lill et al., 2012)) in each dataset was evaluated using epi function. As those SNPs were not present in the lower density Perlgen array used in the Mayo datasets, their best proxies were determined using SNAP in the 1000 genomes CEU population (see below); rs823154 and rs11174928 were used for the analysis in this dataset for PARK16 and LRRK2 respectively. Odd-ratios and their confidence intervals were evaluated using asso function. Meta-analysis and combination of p-values were done using Metal (http://www.sph.umich.edu/csg/abecasis/metal/).

SNP Selection

SNP-SNP pairwise linkage disequilibrium was assessed by SNAP phase (Johnson et al., 2008) using the CEU population panel from the 1000 genomes and HapMap dataset. PD associated SNPs were evaluated based on PDGene meta-analysis results (Lill et al., 2012).

GPI Analysis

Genome-wide SNP variant and gene expression data for 364 individuals were previously described (Myers et al., 2007). Normalized data corrected for covariates such as age, sex and batch effects were processed using R for gene expression analysis and gplink (Purcell et al., 2007) for genotypes. Subsequently for a given SNP, Pearson's correlation coefficient is calculated between the expression level of each gene (within the whole transcriptome dataset) and the allele load across the panel of samples. Associations were arbitrarily described with the high-risk variant at any given disease-associated SNP with positive values, and with the protective low-risk variant with negative values. As a consequence, a gene whose expression is consistently higher in samples from individuals who carry the disease-associated high-risk variant (relatively to the expression in the context of the protective low-risk variant) across the entire sample set will show a positive correlation coefficient (such as Gene 1 in FIG. 12B). By assessing the correlation coefficients across the entire transcriptome for a given variant, the GPI can be obtained.

Formally, the GPI for a SNP is a n-vector of numerical values between −1 and 1, where n is a number of genes whose expression levels is available, and corresponds to the collection of the expression level correlation with the allelic load for each individual gene. The GPI of SNPx was thus calculated as

$\begin{matrix} {{GPISNP}_{x} = \begin{bmatrix} {r\left( {A_{G_{1}},L_{{SNP}_{x}}} \right)} \\ \vdots \\ {r\left( {A_{G_{n}},L_{{SNP}_{x}}} \right)} \end{bmatrix}} & \left. {{{Fig}.\mspace{14mu} 12}C} \right) \end{matrix}$

-   -   with A_(G) _(i) =[(a_(G) _(i) )₁ . . . (a_(G) _(i) )_(p)],         expression level of gene i across all samples, (aGi)_(j) being         the expression level of gene i in sample j     -   L_(SNP) _(x) =[(l_(SNP) _(x) )₁ . . . . (l_(SNP) _(x) )_(p)],         high-risk allele load of SNP x across all samples (l_(SNP)x)         being the high-risk allele load of SNP x in sample j.

$\left( l_{{SNP}_{x}} \right)_{j} = \left\{ \begin{matrix} 0 & {{if}\mspace{14mu} j\mspace{14mu} {is}\mspace{14mu} {homozygous}{\mspace{11mu} \;}{for}\mspace{14mu} {the}\mspace{14mu} {low}\mspace{14mu} {risk}\mspace{14mu} {allele}\mspace{14mu} {of}\mspace{14mu} {SNPx}} \\ 1 & {{if}\mspace{14mu} j\mspace{14mu} {is}\mspace{14mu} {heterozyguous}{\mspace{11mu} \;}{for}\mspace{14mu} {SNPx}} \\ 2 & {{if}\mspace{14mu} j\mspace{14mu} {is}\mspace{14mu} {homozygous}{\mspace{11mu} \;}{for}\mspace{14mu} {the}\mspace{14mu} {high}\mspace{14mu} {risk}\mspace{14mu} {allele}\mspace{14mu} {of}\mspace{14mu} {SNPx}} \end{matrix} \right.$

(See FIG. 12A). (AG1, LSNPx) is the Pearson correlation coefficient between the expression level of gene I and the disease-associated allelic load of SNPx across all samples. (A, LSNPx) is positive for genes whose expression levels are increased in the presence the risk allele and negative for genes whose expression levels are decreased (Genes 1 and 3 respectively in FIG. 12B).

The intersection between the GPIs calculated for two SNPs is evaluated in a threshold-free approach by considering all the genes which show a correlation in the same direction for different GPIs. Formally, with GPI_(SNPxX SNPy)=GPI_(SNPx)∩GPI_(SNPy):

${{GPI}_{{SNP}_{x}{XSNP}_{y}}\lbrack k\rbrack} = \left\{ \begin{matrix} \frac{{{GPI}_{{SNP}_{x}}\lbrack k\rbrack} + {{GPI}_{{SNP}_{y}}\lbrack k\rbrack}}{2} & {{{if}\mspace{14mu} {{GPI}_{{SNP}_{x}}\lbrack k\rbrack}},{{{GPI}_{{SNP}_{y}}\lbrack k\rbrack} > 0}} \\ 0 & {{{if}\mspace{14mu} {{GPI}_{{SNP}_{x}}\lbrack k\rbrack}},{{{GPI}_{{SNP}_{y}}\lbrack k\rbrack} < 0}} \end{matrix} \right.$

In order to study potential interactions between two SNPs, a quantitative trait was defined for which classical genetic interaction analysis could be applied.

A genetic interaction is broadly defined as when the combined phenotypic effect of two mutations (in distinct genes) is not equal to the sum of the two individual phenotypic effects. Thus, such a non-additive interaction can either represent synergy (the combined effect is greater than the sum of its parts) or occlusion (the combined effect is less than the sum of its parts). The prediction for an occlusive genetic interaction is that the transcriptome effect of a risk allele at either one of the 2 genes will preclude the effect of a second risk allele.

A quantitative trait phenotype was defined for the classical genetic interaction analysis. This is most simply done by examining gene expression values that are highly impacted in common by the 2 SNPs individually (as identified above by the GPI intersection genes), and then querying the effect of their combination. Without being bound by theory, any of the gene expression values from the GPI intersection could be queried for a genetic interaction. Rather than querying individual genes expression phenotypes, a single scalar value was generated that represents the combined effect on the expression patterns of all of the relevant genes (as defined by the GPI analysis above; we used the genes most significantly impacted with p<0.01, empirically assessed by resampling). To compute this scalar value, termed the expression quantitative trait (eQT), a standard linear algebra manipulation was used: the combined quantitative trait is the sum of the expression levels of the selected genes, weighted by their GPI intersection coefficients (which reflects how consistently and strongly they are affected by both SNPs).

Formally, the complex expression phenotype for sample j will be:

$\left( {CEP}_{xy} \right)_{j} = {\sum\limits_{m = 1}^{M}\; {{{GPI}_{{SNP}_{x}X\mspace{14mu} {SNP}_{y}}\left\lbrack t_{m} \right\rbrack} \cdot \left( a_{G_{t_{m}}} \right)_{f}}}$

with a selection of M genes from GPI_(SNPxX SNPy) and tm the position in the GPIs vector of the m^(th) highest absolute value in GPI_(SNPxX SNPy).

To actually perform the interaction analysis, we first determine the genotypes for the two SNPs of interest and then proceeded to linear model regression for the quantitative trait across all samples. Computationally, the effect of both SNPs on the quantitative phenotype was assessed using R lm function, by fitting of the linear model CEP_(xy)˜x0+x1·L_(SNPx)+x2·L_(SNPy)+x3·L_(SNPx)L_(SNPy). The test for interaction is based on the significance associated with the coefficient x₃.

Disease Association Expression Profiles

For a given disease dataset, Pearson's correlation coefficient is calculated between the expression level of each gene (within the whole transcriptome dataset) and the disease phenotype across the panel of samples. Associations with the disease were arbitrarily described as positive values, consistently with the orientation assigned in the GPI calculation to the high-risk allele load of a disease-associated SNP. As a consequence, a gene whose expression is consistently higher in disease samples than in unaffected will show a positive correlation coefficient. By assessing the correlation coefficients across the entire transcriptome for a given variant, a global disease profile (GDP) can be obtained.

Formally, the GDP produces an object of the same class as the GPI, a n-vector of numerical values between −1 and 1, where n is a number of genes whose expression levels is available, and corresponds to the collection of the expression level correlation with the disease phenotype (0 for unaffected, 1 for disease) across the samples. Formally, GDPD=

$\quad\begin{bmatrix} {r\left( {A_{G_{1}},P_{D}} \right)} \\ \vdots \\ {r\left( {A_{G_{n}},P_{D}} \right)} \end{bmatrix}$

with PD=[(P_(D))1 . . . (P_(D))p], binary phenotype associated to disease D across all samples, (PD)j being the disease-associated binary phenotype in sample j. (l_(SNPx))_(j)=

$\quad\left\{ \begin{matrix} 0 & {{if}\mspace{14mu} j\mspace{14mu} {is}\mspace{14mu} {unaffected}} \\ 1 & {{if}\mspace{14mu} j\mspace{14mu} {is}\mspace{14mu} {affected}} \end{matrix} \right.$

and r(A_(Gi), P_(D)) the Pearson correlation coefficient between the expression level of gene i and the disease-phenotype across all samples. As a consequence, a gene whose expression level is increase in the course of the disease will be associated to a positive correlation in the disease expression profile. When multiple Affymetrix probesets were available for a given gene, the probeset showing the highest expression level was systematically selected. For resampling procedures, the values of the phenotype vector are randomly reattributed to the different samples (label switch).

Datasets of normalized gene expression (accession numbers GSE20168, GSE7621, GSE1297, GSE3790, GSE12654) were downloaded from the Gene Expression Omnibus website (http://www.ncbi.nlm.nih.gov/geo/).

Similarity between a disease expression profile and a GPI intersection is evaluated by the Pearson correlation between the GPI intersection and the GDP across the subset of genes that show the highest absolute value in the GPI intersection. (In the case of the 7 PD SNPs intersection, the top 135 genes out of the 352 non-null were selected as a core set, with a FDR<5% based on resampling analysis of a 7 SNPs GPI intersection size). All data manipulations and analysis were done using R.

SNP within Probes

Common SNPs within the target sequences of microarray probes have indeed emerged as a potential technical issue for eQTL analysis in recent years (Alberts et al., 2007; Chen et al., 2009), due to the mis-hybridization caused by the allelic variant of such a SNP that does not match the designed target sequence; as a consequence of the poorer hybridization of the probe to its target sequence, the amount of target sequence might be under-evaluated. Classical eQTL studies aim at identifying relationships between the allelic load of a given variant (herein called “studied variant”) and the level of expression of a given gene. In the case of such eQTL studies, results can be biased if the allele leading to a poorer hybridization segregates with one allele of the studied SNP. This most often happens for cis-eQTL, as the local physical structure of the chromosome can lead to a systemic segregation between the studied variant and the one within the probe if those are in linkage disequilibrium. The GPI analysis can be seen as a globalization at a transcriptome-wide scale of eQTL studies, where the effect of a studied variant is considered on transcriptome-wide gene expression levels in a single measurement. As all the genes are considered with the same weight, there are two direct implications to that 1) the global measurement should be robust to potential technical issues (such as SNP-in-Probe) affecting a single probe, as this will only affect a fraction of a percent of the total GPI signal 2) the GPI is mostly based on trans-effect measurements, as more than 99% of the genes will be considered as “trans” by reference to any studied variant.

To empirically support the robustness of the GPI to SNPs in probes, all the probes sequences used in the study were obtained from Illumina, and were mapped on the human genome using Burrows-Wheeler Alignment (Langmead et al., 2009) to identify those that target sequences containing a SNP whose minor allele frequency is superior to 5% in HapMap Caucasian populations (Consortium, 2003). The whole analysis was then reproduced by excluding the 272 probes satisfying those criteria, and the same results were obtained as the one presented in the manuscript in FIGS. 1C and D, establishing the robustness of the GPI procedure, based on its transcriptome-wide design and ruling out any significant effect caused by the presence of SNPs within the probes.

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Example 2 Testing Retromer Drug in LRRK2 Neurite Length Assay

As described herein, the PD gene LRRK2 has been found to affect neurite length. These effects may be mediated by the retromer pathway. A neurite length assay was used to test drug R55, which is reported to stabilize the retromer complex.

Rat primary neurons were transduced at 1 week in culture with vectors expressing the constructs shown in FIG. 14 (along with eGFP for visualization). Thereafter cells were treated either with vehicle or with 5 μM R55. Culture medium was half replaced daily with fresh treated medium for 1 week, and then cells were fixed and examined by fluorescent microscopy. Neurites were traced and measured to find the average total neurite length per neuron in each tested condition.

In vehicle treated cells (black bars in FIG. 14), knocking down VPS35 with shRNA, or overexpressing PD mutant genes VPS35 D620N or LRRK2 G2019S significantly reduced total neurite length compared to vector alone. FIG. 14 also shows for comparison a dominant-negative mutant form of Rac1 (Rac1 DN), which is well-known to cause neurite retraction. (++ p<0.01; +++ p<0.001)

With drug treatment (red bars in FIG. 14), neurons expressing vector alone showed no significant change, while neurons expressing VPS35 shRNA, VPS35 D620N, or LRRK2 G2019S exhibited dramatic rescue of neurite length compared to respective vehicle controls. Neurons expressing Rac1 DN, however, were not affected, suggesting that the drug does not generally protect against neurite retraction. (** p<0.01; *** p≦0.001; n.s. no significant change. n=10-15 neurons per condition. Bars represent mean+SEM. p values calculated by ANOVA with Tukey's HSD post hoc) 

What is claimed is:
 1. A method for treating Parkinson's Disease (PD) in a subject, the method comprising administering to a subject in need thereof a therapeutic amount of a retromer complex-stabilizing compound, or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein the retromer complex-stabilizing compound stabilizes VPS35, VPS29, VPS26 or a combination thereof.
 3. The method of claim 1, wherein the retromer complex-stabilizing compound stabilizes the interaction between VPS35 and VPS29.
 4. The method of claim 1, wherein the retromer complex-stabilizing compound is a compound of formula (I),

wherein, Ar is a 5- or 6-membered aromatic ring or a 5- or 6-membered heteroaromatic ring having 1-4 heteroatoms independently selected from sulfur, nitrogen, or oxygen; X is

or H; R¹ is C₁-C₆-alkyl; A is —S—R², —S(O)R², —SO₂R², —SO₃R²,

and each R² is independently selected from H, C₁-C₆-alkyl, or phenyl.
 5. The method of claim 4, wherein the compound of formula (I) binds to VPS35 and VPS29.
 6. The method of claim 1, wherein the retromer complex-stabilizing compound is R55, wherein the structure of R55 is


7. The method of claim 1, wherein the retromer complex-stabilizing compound is R33, wherein the structure of R33 is 